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TABLE OF CONTENTS

ACRONYMS ................................................................................................................... 3

REGISTRATION REVIEW TEAM................................................................................... 4

PRELIMINARY WORK PLAN ........................................................................................ 5

A. Introduction..................................................................................................... 5

B. Background and Regulatory Information ..................................................... 5

C. Anticipated Risk Assessment and Data Needs ............................................ 6

1. Product Analysis (40 CFR 158.2120)..................................................... 6

2. Human Health Risk Assessment Status (40 CFR 158.2140)................. 8

3. Environmental Fate and Ecological Risk Assessment Status........... 10

4. Risk to Threatened and Endangered Species ..................................... 12

5. Endocrine Effects .................................................................................. 12

6. Incidents................................................................................................. 13

7. Summary of Data Gaps ......................................................................... 13

D. Timeline ......................................................................................................... 13

E. Guidance for Commenters........................................................................... 14

F. Environmental Justice.................................................................................. 14

H. Trade Irritants................................................................................................ 15

I. Additional Information.................................................................................. 15

J. Next Steps ..................................................................................................... 16

BIBLIOGRAPHY: Pseudomonas aureofaciens strain Tx-1 ............................................. 17
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ACRONYMS

Acronym Extension

μg microgram

a.i. Active ingredient

ATTC American Type Tissue Collection

B. cereus Bacillus cereus

CFR Code of Federal Regulations

CFU Colony forming units

DCI Data Call-in

EDSP Endocrine Disruptor Screening Program

EP End-use product

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

EPA Reg. No. EPA Registration Number: a two-part number assigned by EPA to identify each

pesticide product registration.

ETE Emetic Toxin

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FQPA Food Quality Protection Act of 1996

FR Federal Register

g gram

HBL Hemolytic enterotoxin

IDS Incident Data System

L liter

LD50 Median Lethal Dose: a statistically-derived single dose that can be expected to 

cause death in 50% of the test animals when administered by the route indicated 

(oral, dermal, inhalation)

mg/kg bw Milligrams per kilogram of body weight

mg/L Milligrams per liter (approximately equivalent to ppm)

mL Milliliter

MP Manufacturing-use product

MPCA Microbial pesticide control agent

MRID Master Record Identification (number): EPA's system of recording and tracking

studies submitted to the Office of Pesticide Programs.

MRL Maximum residue limits

Nhe Nonhemolytic enterotoxin

NPIC National Pesticides Information Center

OPP EPA’s Office of Pesticide Programs

OPPTS Office of Prevention, Pesticides and Toxic Substances; now the Office of 

Chemical Safety and Pollution Prevention (OCSPP)

PC Code Pesticide Chemical Code: a six-digit number assigned by OPP to identify 

pesticide chemicals.

Ps. Pseudomonas

Syn Synonym

TMDL Total Maximum Daily Load
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REGISTRATION REVIEW TEAM

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Human Health 

John Kough, Ph.D.

Environmental Effects 

Gail Tomimatsu, Ph.D.

Risk Management 

Susanne Cerrelli

Sheryl Reilly, Ph.D.

Office of General Counsel 

Philip Ross, Esq.
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PRELIMINARY WORK PLAN

A. Introduction

The Food Quality Protection Act (FQPA) of 1996 mandated the continuous review of 

existing pesticides. All pesticides distributed or sold in the United States must generally 

be registered by the U.S. Environmental Protection Agency (EPA or the Agency), based 

on scientific data showing that they will not cause unreasonable risks to human health 

or the environment when used as directed on the product labeling. The Registration 

Review program is intended to make sure that, as the ability to assess risk evolves and 

as policies and practices change, all registered pesticides continue to meet the statutory 

standard of no unreasonable adverse effects. Changes in science, public policy, and 

pesticide use practices will occur over time. Through the Registration Review program, 

the Agency periodically reevaluates pesticides to make sure that as change occurs, 

products in the marketplace can continue to be used safely. Information on this program 

is provided at: http://www.epa.gov/oppsrrd1/registration_review/.

The Agency has begun to implement the Registration Review program pursuant to

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) section 3(g) and will review 

each registered pesticide every 15 years to determine whether it continues to meet the 

FIFRA standard for registration. The Agency will consider benefits information and data 

as required by FIFRA. The public phase of Registration Review begins when the initial 

docket is opened for each case. The docket is the Agency’s opportunity to state what it 

knows about the pesticide and what additional risk analyses and data or information it 

believes are needed to make a Registration Review decision. After reviewing and 

responding to comments and data received in the docket during this comment period, 

the Agency will develop and commit to a final work plan and schedule for the 

registration review of Pseudomonas aureofaciens.

This Registration Review Preliminary Work Plan and Summary Document evaluates the 

microbial pest control agent (MPCA) classified under Case 6009: Pseudomonas 

aureofaciens. More information about this case is available in the public docket at 

regulations.gov. To access it, enter the docket number, which is EPA-HQ-OPP-2012-

0421in the blue Search box. 

B. Background and Regulatory Information

Pseudomonas aureofaciens strain Tx-1 (PC Code 006473) was isolated from turf grass 

soil in 1956 at Michigan State University. Pseudomonas aureofaciens (synonym: 

Pseudomonas chlororaphis subspecies aureofaciens) is a naturally occurring bacterium 

that is ubiquitous in soil and plant rhizospheres. Strains of this species have been used 

for biological control of several fungal plant diseases that attack the root zone of various 

plant species. An antibiotic (phenazine) produced by Pseudomonas aureofaciens is 

known to have antifungal properties that are useful in limiting the spread of fungal 

phytopathogens. 
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The only registered pesticide product containing Pseudomonas aureofaciens strain Tx-1 

as a pesticide active ingredient is Spot-less Biofungicide, EPA Reg. No 75801-1, which 

is registered for use for management of turf diseases on golf courses and other turf 

sites. This product was conditionally registered on February 11, 1999. This product is 

unusual in that a trained operator ferments the pesticide product on site in a Bioject 

fermentation system, and performs quality control prior to night-time applications of the 

product to turf grass (including golf courses). The product is applied to turf through

chemigation systems or with conventional sprayers. The Spot-less product label 

contains a use restriction that states that the product is not to be applied to turf or sod 

grown for sale, commercial seed production, and/or research. 

The Pseudomonas aureofaciens species have been reclassified in scientific literature as

Pseudomonas chlororaphis subspecies aureofaciens based on the level of DNA 

similarity of Pseudomonas aureofaciens and Pseudomonas chlororaphis (Johnson and 

Palleroni, 1989; and Palleroni, N.J., 2005), but the registration has not been amended to 

reflect the change in nomenclature for this microorganism. The Registration Review 

case name Pseudomonas aureofaciens reflects the substance name provided when the 

Spot-less product was initially registered. 

Table 1. Summary of Pesticide Product Containing Pseudomonas aureofaciens strain 

Tx-1 (PC Code 006473) as an Active Ingredient

Registration 

No Product Name Registrant First Registered

75801-1

Spot-less 

Biofungicide

Turf Science 

Laboratories, Inc. February 11, 1999

C. Anticipated Risk Assessment and Data Needs

The following summaries describe how the current data requirements for microbial 

pesticides have been addressed for the registration of Pseudomonas aureofaciens

strain Tx-1. The pertinent data areas include: product analysis, residue data, toxicology 

(human health), nontarget organisms, and environmental fate (ecotoxicology). Data 

gaps are also discussed, and are summarized in Table 4. 

1. Product Analysis (40 CFR 158.2120)

At the time of submission, the data were adequate to support the registration of

Pseudomonas aureofaciens strain Tx-1. Additional data are anticipated to be needed, 

however, to ensure that the organism is currently deposited in a nationally recognized 

culture collection (40 CFR 158.2120). The Agency also expects to require the registrant 

to confirm the product identity (including the new species designation, strain, and 

isolate) of the organism if it has not been previously deposited in a nationally recognized 

culture collection. Storage stability, miscibility, and viscosity (40 CFR 158.2120) are 

data requirements that remain outstanding and are expected to be in need of being

addressed during the registration review of this case. 
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This assessment of the product analysis data submitted to support registration is based 

upon adequate quality control as provided at the time of culture of Ps. aureofaciens Tx1 (i.e., when the product is fermented on-site in the BioJect System). The pesticide 

product label and the BioJect® Fermentation and Injection System Operator’s manual 

include quality control measures necessary to assure the integrity of the product.

Table 1. Summary of Product Analysis Data for Pseudomonas aureofaciens strain Tx-1

40 CFR 158.2120 

Data Requirement 

(OPPTS Guideline)

How it Has Been Addressed

MRID(S) EPA Review, Conclusions, Comments)

Product Identity (885.1100) 44337101

44422401

Acceptable

U.S. EPA 1999a

In conjunction with redepositing the Tx1 strain culture in a nationally 

recognized culture collection, the 

Agency anticipates requiring

confirmatory product identity data to 

insure that the characteristics are 

identical to the originally registered 

product.

Manufacturing process 

(885.1200)

44337101

44437501

44461701

Acceptable

U.S. EPA 1999a

Deposition of a sample in a 

nationally recognized culture 

collection (885.1200)

44337101 Additional information is expected to 

be required to confirm that this 

requirement is currently addressed.

Discussion of formation of 

unintentional ingredients 

(885.1300)

44337101

44461701

Acceptable

U.S. EPA 1999a

Analysis of samples (885.1400) 44337102

45048501

45077601

Acceptable 

U.S. EPA 2003

Certification of limits (885.1500) CSF dated 

44337102

Additional data are expected to be 

required. If the nomenclature of the 

active ingredient is amended or a cell 

deposit number is submitted, a new 

CSF will need to be submitted that 

reflects the change(s).

Color (830.6302) 44337103 Orange 

Acceptable

Physical state (830.6303) 44337103 Liquid 

Acceptable

Odor (830.6304) 44337103 Slightly musty

Acceptable
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40 CFR 158.2120 

Data Requirement 

(OPPTS Guideline)

How it Has Been Addressed

MRID(S) EPA Review, Conclusions, Comments)

pH (830.7000) 44337103 7.5

Acceptable

Density/relative density/bulk 

density (specific gravity) 

(830.7300)

44337103 9.1 lbs. /gal. 

Acceptable

Corrosion characteristics 

(830.6320)

44337103 Noncorrosive to plastic 

Acceptable

Storage stability (830.6317) These data are expected to be required.

Stability to normal and elevated 

temperatures, metals and metal 

ions (830.6313)

44337103 Stable in water at normal and elevated 

temperature (85 ̊F max). Not stable if 

stored in direct sunlight. Acceptable

Miscibility (830.6319) These data are expected to be required for

Spot-less Biofungicide.

Viscosity (830.7100) These data are expected to be required for

Spot-less Biofungicide.

NON-GUIDELINE- Literature 

Review For Pseudomonas 

produced antibiotics

45077601 Acceptable. U.S EPA 2003

2. Human Health Risk Assessment Status (40 CFR 158.2140)

All mammalian toxicology data requirements have been satisfied. During initial 

registration, EPA determined that there would be no human health concerns arising 

from the use of Pseudomonas aureofaciens strain Tx-1 in pesticide products, and 

provided a public interest finding in support of pesticides containing Pseudomonas 

aureofaciens strain Tx-1 as an alternative to more toxic conventional pesticides for turf 

disease control (U.S. EPA, 1999a.)

The acute oral toxicity and dermal irritation studies submitted for the active ingredient 

Ps. aureofaciens Tx-1 demonstrate a lack of toxicity or adverse effects following dosage 

with amounts (expressed as colony forming units or “cfu”) surpassing those that are 

present following application of this bacterium through irrigation lines and sprinklers on 

golf courses. This determination is for the active ingredient as applied through the 

BioJect Automated Microbial Fermentation and Injection System as currently designed; 

any changes in the identity of the active ingredient or alterations to the BioJect System 

will require a full assessment for potential adverse effects.

The human health toxicity and pathogenicity data are summarized in the following table:
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Table 2. Summary of Toxicology Data for Pseudomonas aureofaciens strain Tx-1

40 CFR 158.2140 

Data Requirement 

(OPPTS Guideline)

How it Has Been Addressed

MRID EPA Review, Conclusions , Comments

Acute oral toxicity/ 

pathogenicity 

(885.3050)

40384802 Data were bridged from test material Ps. fluorescens

EG-1053-TGAI.

There was no significant effect on mean body weight 

gain, and no mortality associated with treatment. Not 

pathogenic at 2.6 x 109cfu/kg of body weight. Toxicity 

Category IV. Classification: Acceptable. U.S. EPA 

1999a

Acute pulmonary 

toxicity/pathogenicity 

(885.3150)

40384806

Data were bridged from test material Ps. fluorescens

EG-1053-TGAI.

Ps. fluorescens was not infective, toxic, nor 

pathogenic to rats when administered at 1.5 x 105

cfu/animal. There was no effect on mean body weight 

gain, no mortality, and no treatment-related lesions or 

other adverse effects. Toxicity Category IV.

Classification: Acceptable U.S. EPA 1999a

Acute injection 

toxicity/ 

pathogenicity—

intravenous 

(885.3200)

40384808 Data were bridged from test material Pseudomonas

fluorescens EG-1053-TGAI.

There was no significant effect on mean body weight 

gain, no mortality, and no treatment related lesions

when administered at 2.2 x 108cfu/kg.

Toxicity Category IV. Classification: Acceptable

U.S. EPA 1999a

Cell culture 

(885.3500)

Not Applicable. Ps. aureofaciens is not a virus (refer to test note 

#4 of 40 CFR § 158.2140(d)).

Hypersensitivity 

incidents (885.3400)

Not 

Applicable

No hypersensitivity incidents occurring during the 

testing, production, or use of Ps. aureofaciens were 

reported by the registrant. Any future 

hypersensitivity incidents must be reported (refer to 

test note #3 of 40 CFR § 158.2140(d)).

Acute oral 

toxicity (870.1100)

44337104 One female rat displayed weight loss from day 7 to 

day14; and dilated pupils were noted in 3 female 

rats on days 1 to 4. No Abnormalities noted at 

necropsy. LD50 > 5000 mg/kg of BW.

Toxicity Category IV. U.S. EPA 1999a

Acute dermal

toxicity (870.1200)

40384804 Data were bridged from test material Ps.

fluorescens EG-1053-TGAI. Mean body weights 

(rats) increased during the 14 day study; Ps. 

fluorescens was considered non-toxic when applied 

to rabbit skin. Toxicity Category IV

U.S. EPA 1999a
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40 CFR 158.2140 

Data Requirement 

(OPPTS Guideline)

How it Has Been Addressed

MRID EPA Review, Conclusions , Comments

Acute inhalation

toxicity (870.1300)

Not applicable to the TGAI test substance.

Acute eye irritation 

(870.2400)

Not applicable to the TGAI test substance.

Primary dermal

irritation (870.2500)

44337105 Slight dermal irritation and erythema at 1 hour; 

resolved by 72 hours. Tan discoloration at test site 

in all rabbits. Primary irritation index=0.63

Toxicity Category IV. Classification: Acceptable

U.S. EPA 1999a

3. Environmental Fate and Ecological Risk Assessment Status

(40 CFR 158.2150)

The ecotoxicity data which satisfy Tier I data requirements are summarized in Table 3 

below. No data gaps have been identified.

Table 3. Ecological Toxicity Data for Pseudomonas aureofaciens strain Tx-1

40 CFR 158.2150 

Data Requirement 

(OPPTS Guideline)

How it Has Been Addressed

MRID EPA Review, Conclusions, Comments

Avian oral toxicity 

(885.4050)

45040804

45040805

Pseudomonas aureofaciens strain Tx-1 is practically 

nontoxic tand is not pathogenic to Mallard Duck and 

Bob White Quail (NOEL= 5 x109 CFU/kg/day )

Classification: Acceptable

U.S. EPA 2008b and c

Avian inhalation 

toxicity/pathogenicity 

(885.4100)

Not 

applicable

No testing expected to be required. Results of 

acceptable oral toxicity studies determined that 

Pseudomonas aureofaciens strain Tx-1 is practically 

nontoxic and not pathogenic to avian species.

Wild mammal toxicity/ 

pathogenicity 

(885.4150)

Not 

applicable

No testing expected to be required. Results of the

Tier 1 toxicity tests required by 40 CFR § 158.2140 

(Table 2) are adequate and appropriate for 

assessment of hazards 

Freshwater fish 

toxicity/

pathogenicity 

(885.4200)

45040801

45040803

Pseudomonas aureofaciens strain Tx-1 is not 

infectious and practically nontoxic to 

both coldwater and warm water fish species. The 30 

day LC50 is > 2.2 x107 CFU/ml for rainbow trout and 

>3.4 x 105for blue gill. The NOEC for rainbow trout 

and blue gill is 9.3 x105 CFU/ml.

Classification: Acceptable
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40 CFR 158.2150 

Data Requirement 

(OPPTS Guideline)

How it Has Been Addressed

MRID EPA Review, Conclusions, Comments

U.S. EPA 2007a. and U.S. EPA 2008a.

Freshwater 

invertebrate toxicity/ 

pathogenicity 

(885.4240)

45040802 Pseudomonas aureofaciens strain Tx-1 is not 

infectious and practically nontoxic to

Daphnia magna at 1.7 x 104 CFU/ml.

Classification: Acceptable

U.S. EPA 2007b.

Estuarine/Marine fish 

testing

Estuarine and marine 

invertebrate testing 

(885.4280)

Not 

applicable

No testing expected to be required. Significant 

exposures to estuarine/marine organisms are not 

expected organisms are not expected as a result of 

application to golf courses. Also, Pseudomonas 

aureofaciens strain Tx-1 is not infectious and 

practically nontoxic to 

freshwater fish and aquatic invertebrate species

Nontarget plant 

testing (885.4300)

Not 

applicable

No testing expected to be required, if exposures to 

Nontarget plants are limited to those species on turf 

and golf courses.

U.S. EPA 1998.

Nontarget insect 

testing (885.4340)

45265201 For green lace wings the LC50 is >8.5 x 106 CFU/g 

and the NOEC is 8.5 x 106 CFU/g.

Classification: Acceptable

U.S. EPA 2002.

Nontarget insect 

testing (885.4340)

45225201 For Ladybird Beetles the dietary LC50 is >4.28 x107

CFU/g and the NOEC is 4.28 x107 CFU/g.

Classification: Acceptable

U.S. EPA 2001.

Nontarget insect 

testing (885.4340)

45225202 For parasitic Hymenoptera the dietary LC50 is >4.28 x 

107 CFU/g and the NOEC is 4.28 x107 CFU/g.

Classification: Acceptable

U.S. EPA 2001.

Honey bee testing 

(885.4380)

45077602 The reviewed data indicate Pseudomonas 

aureofaciens strain Tx-1 is not toxic at 106- 107

CFU/ml, or pathogenic at 103 CFU/ml. In addition, 

Pseudomonas aureofaciens has never been reported 

as pathogenic to bees.

Classification: Acceptable

US EPA 2000.
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4. Risk to Threatened and Endangered Species

Based on the specific use patterns and the absence of data gaps in ecological data, the 

Agency does not anticipate the need for new data for Pseudomonas aureofaciens strain 

Tx-1. Because this a.i. has a product that is registered for outdoor use, the Agency is 

required to consider any effects on nontarget species, particularly those with 

endangered or threatened status. 

EPA has not conducted a risk assessment that supports a complete endangered 

species determination for Pseudomonas aureofaciens strain Tx-1. The ecological risk 

assessment planned during Registration Review will allow the Agency to determine 

whether use of Pseudomonas aureofaciens strain Tx-1 will have “no effect” or “may 

affect” federally-listed threatened or endangered species (listed species) or their 

designated critical habitat. When an assessment concludes that a pesticide’s use “may 

affect” a listed species or its designated critical habitat, the Agency will consult with the 

U.S. Fish and Wildlife Service and/or National Marine Fisheries Service (the Services), 

as appropriate.

5. Endocrine Effects

As required by the Administrator under the Federal Food, Drug, and Cosmetic Act 

(FFDCA) section 408(p), EPA has developed the Endocrine Disruptor Screening

Program (EDSP) and has begun to implement the screening program that is to be used

to test all pesticides in order 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.” 

FFDCA section 408(p)(4), authorizes the Administrator, by order, to exempt from the 

requirements of the Estrogenic Substances Screening Program a biologic substance or 

other substance if a determination is made that the substance is not anticipated to 

produce any effect in humans similar to an effect produced by a naturally occurring 

estrogenic substance.

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. Pseudomonas aureofaciens strain Tx-1 is not among the group of 58 

pesticide active ingredients on the initial list to be screened under the EDSP. 

The Agency believes that the active ingredient involved in this Registration Review case

likely is a substance that would not produce any effect in humans similar to an effect 

produced by a naturally occurring estrogenic substance. As such, pursuant to Section 

408(p)(4), EPA will determine if it can exempt Pseudomonas aureofaciens strain Tx1from the requirements of the Section 408(p) EDSP. If the Agency determines that it is 

appropriate to exempt this active ingredient from EDSP screening, an order will be 

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

of 67 chemicals, future lists, the test guidelines and the Tier 1 screening battery, please 

visit our website: http://www.epa.gov/endo/.

6. Incidents

The National Pesticides Information Center (NPIC) database reported no incidents 

associated with products containing Pseudomonas aureofaciens strain Tx-1 use. The 

Office of Pesticide Programs’ Incident Data System (IDS) indicates that there have been 

no reports of adverse human, domestic animal, or environmental incidents for 

Pseudomonas aureofaciens strain Tx-1 products.

7. Summary of Anticipated Data Needs

Table 4 includes anticipated data needs for Pseudomonas aureofaciens. For 

additional discussion of the anticipated data needs, see section C. 1. Product Analysis 

above.

Anticipated Data Needs for Registration Review of Pseudomonas aureofaciens 

Guideline 

Number Study Title

Test Material

Estimated 

Timeframe

(Months from 

receipt of 

DCI)

885.1100 Product Identity1EP 12

Deposition of a sample in a nationally 

recognized culture collection TGAI 12

885.1500 Certification of limits EP 12

830.6317 Storage stability TGAI and EP 12

830.6319 Miscibility EP 12

830.7100 Viscosity EP 12

TGAI = technical grade active ingredient; EP = end-use product

1

In conjunction with redepositing the Tx-1 strain culture in a nationally recognized culture collection, the 

Agency anticipates requiring confirmatory product identity data to insure that the characteristics are 

identical to the originally registered product.

D. Timeline

The projected timeline for Case 6009— Pseudomonas aureofaciens strain Tx-1 is listed 

in table 5, below. This may be revised, if more time is needed (e.g., issuance of a DCI

ESA Consultation with Fish and Wildlife Services, etc.), and included in the Final Work 

Plan.
-14-

Table 5. Projected Timeline for the Registration Review of Case 6009

Activity Estimated Month

and Year

Phase 1: Open the Public Docket

Open Comment Period for Docket June 30, 2012

Close Comment Period August 30, 2012

Phase 2: Case Development

Develop Final Work Plan January 2013

Issue Data Call-in (DCI) January 2014

Data Submission January 2016

Open Public Comment Period for Preliminary Risk Assessments July 2017

Close Public Comment Period August 2017

Phase 3: Registration Review Decision

Open Comment Period for Proposed Registration Review Decision January 2018

Close Comment Period March 2018

Final Decision and Begin Post-Decision Follow-up 2018

Estimated Total (years) 6

E. Guidance for Commenters

The public is invited to comment on EPA’s preliminary work plan and rationale. The 

Agency will consider all comments as well as any additional information or data 

provided in a timely manner prior to issuing a final work plan for this case.

F. Environmental Justice

EPA seeks to achieve environmental justice, the fair treatment and meaningful

involvement of all people, regardless of race, color, national origin, or income, in the

development, implementation, and enforcement of environmental laws, regulations, and

policies. To help address potential environmental justice issues, the Agency seeks 

information on any groups or segments of the population who, as a result of their 

location, cultural practices, or other factors, may have atypical, unusually high exposure 

to Pseudomonas aureofaciens strain Tx-1 compared to the general population. Please 

comment if you are aware of any subpopulations that may have atypical, unusually high 

exposure compared to the general population.

G. Water Quality

Pseudomonas aureofaciens strain Tx-1 is not identified as a cause of impairment for 

any water bodies listed as impaired under section 303(d) of the Clean Water Act, based 

on information provided at: http://www.epa.gov/owow/tmdl/. In addition, no Total 
-15-

Maximum Daily Loads (TMDL) have been developed for Pseudomonas aureofaciens

strain Tx-1 based on information provided at: 

http://iaspub.epa.gov/tmdl_waters10/text_search.tmdl_search_form

More information on impaired water bodies and TMDL’s can be found at: 

http://www.epa.gov/owow/tmdl/ 

The Agency invites submission of water quality data for this pesticide. To the extent 

possible, data should conform to the quality standards in Appendix A of the Office of 

Pesticide Program’s (OPP) Standard Operating Procedure: Inclusion of Impaired Water 

Body and Other Water Quality Data in OPP’s Registration Review Risk Assessment and 

Management Process to ensure they can be used quantitatively or qualitatively in 

pesticide risk assessments. This information can be located at:

http://www.epa.gov/oppsrrd1/registration_review/water_quality_sop.htm

H. Trade Irritants

Through the Registration Review process, the Agency intends to solicit information on

trade irritants and, to the extent feasible, take steps toward facilitating irritant resolution. 

Growers and other stakeholders are asked to comment on any trade irritant issues 

resulting from lack of Maximum Residue Limits (MRL’s) or disparities between United 

States tolerances and MRL’s in key export markets, providing as much specificity as 

possible regarding the nature of the concern.

Pseudomonas aureofaciens strain Tx-1 active ingredient is not applied to food or feed in 

the United States. In addition, there are no known MRL’s (FAO and WHO 2010). 

Therefore, the Agency does not anticipate that current uses of this active ingredient will 

pose concerns as trade irritants. 

I. Additional Information

Stakeholders are specifically asked to provide information and data that will assist the 

Agency in refining the risk assessments, including any species-specific ecological 

effects determinations. The Agency is interested in obtaining the following information 

regarding the use of Pseudomonas aureofaciens strain Tx-1: 

1. Confirmation on the following label information:

a. Sites of application

b. Formulations

c. Application methods and equipment

d. Maximum application rates
-16-

e. Frequency of application, application intervals and maximum 

number of applications

f. Geographic limitations on use

2. Use or potential use distribution

3. Use history

4. Usage/use information for nonagricultural uses (e.g., materials 

preservation)

5. Typical application interval

6. State or local use restrictions

7. Monitoring data

J. Next Steps

After the 60-day comment period closes, the Agency will review and respond to any 

comments received in a timely manner, then issue a Final Work Plan for Case 6009: 

Pseudomonas aureofaciens.
-17-

BIBLIOGRAPHY: Pseudomonas aureofaciens strain Tx-1

MRID Studies

44337100 EcoSoil Systems Inc. (1997) Submission of Product Chemistry and Toxicity 

Data in Support of the Application for Registration of Ping Fungicide. 

Transmittal of 6 Studies. 

44337101 Harrison, E. (1997) Ping Fungicide: Product Identity and Composition. 

Unpublished study prepared by EcoSoil Systems, Inc. 81 p. 

44337102 Harrison, E. (1997) Ping Fungicide: Analysis and Certification of Product 

Ingredients. Unpublished study prepared by Encore Technologies. 12 p. 

44337103 Harrison, E. (1997) Ping Fungicide: Physical and Chemical Properties. 

Unpublished study prepared by EcoSoil Systems, Inc. 4 p. 

44337104 Douds, D. (1997) An Acute Oral Toxicity Study in Rats with Pseudomonas 

aureofaciens Strain Tx-1: Final Report: Lab Project Number: 3446.1. 

Unpublished study prepared by Springborn Laboratories, Inc. 23 p. 

44337105 Douds, D. (1997) A Primary Skin Irritation Study in Rabbits with Pseudomonas 

aureofaciens Strain Tx-1: Final Report: Lab Project Number: 3446.2. 

Unpublished study prepared by Springborn Laboratories, Inc. 29 p. 

44337106 Harrison, E. (1997) Justification for Using Data on P. fluorescens to Satisfy 

Data Requirements for P. aureofaciens. Unpublished study prepared by EcoSoil 

Systems, Inc. 169 p. 

44422400 Eco Soil Systems (1997) Submission of Product Chemistry and Environmental 

Fate Data in Support of the Registration of the Biopesticide based on the Active 

Ingredient Pseudomonas aureofaciens strain Tx-1. Transmittal of 1 Study. 

44422401 Debus, T.; Odelson, D. (1997) Pseudomonas aureofaciens strain Tx-1: 

Supplemental Product Identification Data to Support Registration: Lab Project 

Number: SC 3867. Unpublished study prepared by Eco Soil Systems, Inc. 17 p. 

44437500 Eco Soil Systems, Inc. (1997) Submission of Product Chemistry Data in Support 

of the Application for Registration of Biopesticide Pseudomonas aureofaciens 

strain Tx-1. Transmittal of 1 Study. 

44437501 Ourisson, P. (1997) Growth of Pseudomonas aureofaciens Strain Tx-1 and Other 

Microorganisms Using the BioJect Automated Microbial Fermentation and 

Injection System (Bioject): Lab Project Number: 245-502. Unpublished study 

prepared by Quality Associates, Inc. 87 p. 

44461700 Eco Soil Systems, Inc. (1998) Submission of Toxicity, Product Chemistry and 

Hazard Evaluation Data in Support of the Application for Registration of Spot-
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Less Biofungicide. Transmittal of 3 Studies. 

44461701 Odelson, D.; Debus, T. (1998) Spot-Less Biofungicide: Response to EPA Letter 

of December 17, 1997: Manufacturing Process and Discussion of Formation of 

Impurities. Unpublished study prepared by Eco Soil Systems, Inc. 12 p. 

{OPPTS 885.1200 and 885.1300} 

44461702 Odelson, D.; Debus, T. (1998) Spot-Less Biofungicide: Response to EPA Letter 

of December 17, 1997: Information to Satisfy Acute Toxicity Data 

Requirements. Unpublished study prepared by Eco Soil Systems, Inc. 6 p. 

44461703 Odelson, D.; Debus, T. (1998) Spot-Less Biofungicide: Response to EPA Letter 

of December 17, 1997: Literature Cross References Supporting the Citation of 

Data Conducted on Pseudomonas fluorescens. Unpublished study prepared by 

Eco Soil Systems, Inc. 103 p. 

44708700 Eco Soil Systems (1998) Submission of Efficacy Data in Support of the 

Application for Registration of Spot-Less Biofungicide. Transmittal of 1 Study. 

44708701 Odelson, D. (1998) Addition of Non-Target Bacteria and Pseudomonas 

aureofaciens Strain Tx-1 into the BioJect Automated Microbial Fermentation 

and Injection System (BioJect): Lab Project Number: ESSI-109801. 

Unpublished study prepared by Eco Soil Systems. 31 p. 

44713300 Eco Soil Systems, Inc. (1998) Submission of Efficacy Data in Support of the 

Application for Registration of Spot-Less Biofungicide. Transmittal of 1 Study. 

44713301 Dwyer, P.; Vargas, J.; Bresnahan, J. et al. (1998) Compilation of Efficacy Data 

for the Microbial Pesticide Pseudomonas aureofaciens Strain Tx-1: (Spot-Less). 

Unpublished study prepared by Eco Soil Systems. 23 p. 

45040800 Eco Soil Systems, Inc. (2000) Submission of Toxicity Data in Support of the 

Registration of Spot-Less Biofungicide. Transmittal of 5 Studies. 

45040801 Drottar, K.; Krueger, H. (2000) Pseudomonas aureofaciens Strain TX-1 

Biopesticide: A Five Concentration Toxicity and Pathogenicity Test with the 

Rainbow Trout (Oncorhynchus mykiss) Final Report: Lab Project Number: 

479A-103. Unpublished study prepared by Wildlife International, Ltd. 30 p. 

{OPPTS 885.4200} 

45040802 Drottar, K.; Krueger, H. (2000) Pseudomonas aureofaciens Strain TX-1 

Biopesticide: A 21-Day Life-Cycle Toxicity and Pathogenicity Test with the 

Cladoceran (Daphnia magna): Final Report: Lab Project Number: 479A-101A. 

Unpublished study prepared by Wildlife International, Ltd. 35 p. {OPPTS 

885.4250} 

45040803 Drottar, K.; Krueger, H. (2000) Pseudomonas aureofaciens Strain TX-1 

Biopesticide: A Five Concentration Toxicity and Pathogenicity Test with the 

Bluegill (Lepomis macrochirus): Final Report: Lab Project Number: 479A-102. 

Unpublished study prepared by Wildlife International, Ltd. 30 p. {OPPTS 

885.4200} 
-19-

45040804 Gallagher, S.; Casey, C.; Beavers, J. (2000) Pseudomonas aureofaciens Strain 

TX-1 Biopesticide: An Avian Oral Pathogenicity and Toxicity Study in the 

Northern Bobwhite: Lab Project Number: 479-100. Unpublished study prepared 

by Wildlife International, Ltd. 32 p. {OPPTS 885.4050} 

45040805 Gallagher, S.; Casey, C.; Beavers, J. (2000) Pseudomonas aureofaciens Strain 

TX-1 Biopesticide: An Avian Oral Pathogenicity and Toxicity Study in the 

Mallard: Lab Project Number: 479-102. Unpublished study prepared by Wildlife 

International, Ltd. 32 p. 

45048500 Eco Soil Systems, Inc. (2000) Submission of Product Chemistry Data in Support 

of the Registration of Spot-Less Biofungicide. Transmittal of 1 Study. 

45048501 Odelson, D. (2000) Analysis of Bioject Grown Pseudomonas aureofaciens 

Strain Tx-1 (Spot-Less Biofungicide) for Antibiotics. Unpublished study 

prepared by U.S. Dept. of Agriculture. 16 p. {OPPTS 885.1400} 

45077600 Eco-Soil Systems, Inc. (2000) Submission of Toxicity and Efficacy Data in

Support of the Registration of Spot-Less Biofungicide. Transmittal of 2 Studies. 

45077601 Swick, C. (2000) Review of Antibiotic Production by Pseudomonads. 

Unpublished study prepared by Eco Soil Systems, Inc. 265 p. 

45077602 Odelson, D. (2000) Evaluation of Dietary Effect(s) of Pseudomonas 

aureofaciens Strain TX-1 (Spotless) on Adult Honey Bees (Apis mellifera L.): 

Lab Project Number: CAR 184-99. Unpublished study prepared by California 

Agricultural Research, Inc. 101 p. {OPPTS 885.4340} 

45225200 Eco Soil Systems, Inc. (2000) Submission of Toxicity Data in Support of the 

Registration of Spot-Less Biofungicide. Transmittal of 2 Studies. 

45225201 Bryan, R.; Porch, J.; Krueger, H. (2000) Pseudomonas Aureofaciens Strain TX1: A Dietary Pathogenicity and Toxicity Study with the Ladybird Beetle 

(Hippodamia convergens): Final Report: Lab Project Number: 479-103B. 

Unpublished study prepared by Wildlife International, Ltd. 20 p. 

45225202 Bryan, R.; Porch, J.; Krueger, H. (2000) Pseudomonas aureofaciens Strain TX1: A Dietary Pathogenicity and Toxicity Study with Parasitic Hymenoptera 

(Nasonia vitripennis): Final Report: Lab Project Number: 479-104B. 

Unpublished study prepared by Wildlife International, Ltd. 21 p. 

45265200 Eco Soil Systems, Inc. (2000) Submission of Toxicity Data in Support of the 

Registration of Spot-Less Biofungicide. Transmittal of 1 Study. 

45265201 Bryan, R.; Porch, J.; Krueger, H. (2000) Pseudomonas aureofaciens strain TX-1: 

A Dietary Pathogenicity and Toxicity Study with Green Lacewing Larvae 

(Chrysoperla carnea): Lab Project Number: 479-105A. Unpublished study 

prepared by Wildlife International, Ltd. 17 p. {OPPTS 885.4340} 
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EPA Reviews

U.S. EPA 1998. Memorandum from Gail Tomimatsu, Ph.D. to Michael Mendelsohn. 

Eco Soil System's Request to Bridge Ecotoxicity Studies from Pseudomonas

fluorescens (Dagger G. Biofungicide, EPA Reg. No. 55638-5) to a New Microbial Active 

Ingredient, Pseudomonas aureofaciens Strain Tx-1; DP Barcode: D244137; Case No. 

061928. June 29, 1998.

U.S. EPA 1999a. Registration Eligibility Decision: Pseudomonas aureofaciens Strain 

Tx-1. Environmental Protection Agency, Office of Pesticide Programs. 

February 11, 1999.

U.S. EPA 1999b. Memorandum from Chris Wozniak, Ph.D to Michael Mendelsohn.

Assessment of Pseudomonas aureofaciens Strain Tx-1, Spot-Less, a Biological 

Fungicide, as Applied Through Boom-type Sprayers. Environmental Protection Agency,

Office of Pesticide Programs. June 29, 1999.

U.S. EPA 2000. Memorandum from Robyn Rose to Michael Mendelsohn. Review of 

study designed to evaluate the dietary effects of adult honey bees feeding on

Pseudomonas aureofaciens strain TX-1 (SpotlessTM) for EPA Reg. No. 70688-1 U.S. 

Environmental Protection Agency, Office of Pesticide Programs. June 12, 2000.

U.S. EPA 2001. Memorandum from Robyn Rose to Michael Mendelsohn. Review of 

ecological studies including Dietary Pathogenicity/Toxicity for lady beetles, green 

lacewing larvae, and parasitic Hymenoptera for Pseudomonas aureofaciens strain TX-1. 

EPA Reg. No. 70688-1; (MRID Nos 452252-01, 452652-01, and 452252-02). U.S. 

Environmental Protection Agency, Office of Pesticide Programs. August 9, 2001

U.S. EPA 2002. Memorandum from Robyn Rose to Susanne Cerrelli. Review of Eco 

Soil Systems, Inc. response to BPPD comments on green lacewing studies addressed 

in the April 9, 2001 review of Pseudomonas aureofaciens strain TX-I. EPA Reg. No. 

70688-1 (MRID No 452652-01). U.S. Environmental Protection Agency, Office of 

Pesticide Programs. February 4, 2002.

U.S. EPA 2003. Memorandum from Joel Gagliardi, Ph.D. to Susanne Cerrelli. Review of

Five-batch analysis including a production study, and background literature, to support

unconditional registration of Spot-less Biofungicide. November 21, 2003.

U.S. EPA 2007a., Russell S. Jones, Ph.D. and Gail S. Tomimatsu, Ph.D. Data 

Evaluation Report of Pseudomonas aureofaciens Strain TX-1 Biopesticide: A FiveConcentration Toxicity and Pathogenicity Test with the Rainbow Trout (Oncorhynchus 

mykiss). (MRID # 450408-01) U.S. Environmental Protection Agency, Office of 

Pesticide Programs. November 6, 2007.
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U.S. EPA 2007b., Russell S. Jones, Ph.D. and Gail S. Tomimatsu, Ph.D. Data 

Evaluation Report of Pseudomonas aureofaciens Strain TX-1 Biopesticide : A 21-Day 

Life-Cycle Toxicity and Pathogenicity Test with the Cladoceran (Daphnia magna) (MRID 

# 450408-02.) U.S. Environmental Protection Agency, Office of Pesticide Programs. 

November 6, 2007.

U.S. EPA 2008a., Russell S. Jones, Ph.D. and Gail S. Tomimatsu, Ph.D. Data 

Evaluation Report of Pseudomonas aureofaciens Strain TX-1 Biopesticide: A FiveConcentration Toxicity and Pathogenicity Test with the Bluegill (Lepomis macrochirus) 

(MRID # 450408-03) U.S. Environmental Protection Agency, Office of Pesticide 

Programs. January 10, 2008.

U.S. EPA 2008b. Russell S. Jones, Ph.D. and Gail S. Tomimatsu, Ph.D. Data 

Evaluation Report of Pseudomonas aureofaciens Strain TX-1: An Avian Oral 

Pathogenicity and Toxicity Study in Northern Bobwhite [MRID # 450408-04]. U.S. 

Environmental Protection Agency, Office of Pesticide Programs. January 10, 2008. 

U.S. EPA 2008c., Russell S. Jones, Ph.D. and Gail S. Tomimatsu, Ph.D. Data 

Evaluation Report of Pseudomonas aureofaciens Strain TX-1: An Avian Oral 

Pathogenicity and Toxicity Study in Mallard (MRID # 450408-05). U.S. Environmental 

Protection Agency, Office of Pesticide Programs. January 16, 2008.

Other Information

Palleroni, N.J., 2005. Genus I. Pseudomonas, pp. 351-358; 362-363 in: Brenner, D. J., 

Krieg, N. R. and Staley, J.T. Bergey’s Manual of Systematic Bacteriology, Volume 2, 2nd

Ed. Springer, New York.

Johnson and Palleroni, 1989. “Deoxyribonucleic Acid Similarities among Pseudomonas 

Species.” International Journal of Systematic Bacteriology, Vol. 39. No. 3 (July 1989), 

230-235.