BIOPESTICIDES REGISTRATION ACTION DOCUMENT

Trichoderma asperellum strain ICC 012

PC Code: 119208

U.S. Environmental Protection Agency

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

Last Updated – December 23, 2009

Table of Contents

  TOC \o "1-3" \h \z \u    HYPERLINK \l "_Toc249342512"  I.  EXECUTIVE
SUMMARY	  PAGEREF _Toc249342512 \h  4

  HYPERLINK \l "_Toc249342513"  II.  ACTIVE INGREDIENT OVERVIEW
PAGEREF _Toc249342513 \h  6

  HYPERLINK \l "_Toc249342514"  III.  REGULATORY BACKGROUND	  PAGEREF
_Toc249342514 \h  6

  HYPERLINK \l "_Toc249342515"  IV.  RISK ASSESSMENT	  PAGEREF
_Toc249342515 \h  7

  HYPERLINK \l "_Toc249342516"  A.  Product Analysis Assessment
PAGEREF _Toc249342516 \h  8

  HYPERLINK \l "_Toc249342517"  1.  Product Chemistry and Composition
PAGEREF _Toc249342517 \h  8

  HYPERLINK \l "_Toc249342518"  2.  Analysis and Certified Limits
PAGEREF _Toc249342518 \h  8

  HYPERLINK \l "_Toc249342519"  3.  Physical and Chemical
Characteristics	  PAGEREF _Toc249342519 \h  9

  HYPERLINK \l "_Toc249342520"  B.  Human Health Assessment	  PAGEREF
_Toc249342520 \h  9

  HYPERLINK \l "_Toc249342521"  1.  Toxicology	  PAGEREF _Toc249342521
\h  9

  HYPERLINK \l "_Toc249342522"  2.  Dietary Exposure and Risk
Characterization	  PAGEREF _Toc249342522 \h  13

  HYPERLINK \l "_Toc249342523"  3.   Drinking Water Exposure and Risk
Characterization	  PAGEREF _Toc249342523 \h  13

  HYPERLINK \l "_Toc249342524"  4.  Acute and Chronic Dietary Exposure
and Risks for Sensitive	  PAGEREF _Toc249342524 \h  13

  HYPERLINK \l "_Toc249342525"  Subpopulations, Particularly Infants and
Children	  PAGEREF _Toc249342525 \h  13

  HYPERLINK \l "_Toc249342526"  Characterization	  PAGEREF _Toc249342526
\h  14

  HYPERLINK \l "_Toc249342527"  6.  Aggregate Exposure from Multiple
Routes Including Dermal, Oral, and	  PAGEREF _Toc249342527 \h  14

  HYPERLINK \l "_Toc249342528"  Inhalation	  PAGEREF _Toc249342528 \h
14

  HYPERLINK \l "_Toc249342529"  7.  Cumulative Effects	  PAGEREF
_Toc249342529 \h  15

  HYPERLINK \l "_Toc249342530"  8.  Risk Characterization	  PAGEREF
_Toc249342530 \h  15

  HYPERLINK \l "_Toc249342531"  C.  Environmental Assessment	  PAGEREF
_Toc249342531 \h  15

  HYPERLINK \l "_Toc249342532"  1.  Summary of Non-target Organism
Testing and Waiver Rationales	  PAGEREF _Toc249342532 \h  15

  HYPERLINK \l "_Toc249342533"  2.  Environmental Effects Conclusions
PAGEREF _Toc249342533 \h  19

  HYPERLINK \l "_Toc249342534"  3. Threatened and Endangered Species
Assessment	  PAGEREF _Toc249342534 \h  22

  HYPERLINK \l "_Toc249342535"  V.	ENVIRONMENTAL JUSTICE	  PAGEREF
_Toc249342535 \h  23

  HYPERLINK \l "_Toc249342536"  VI.   RISK MANAGEMENT AND REGISTRATION
DECISIONS	  PAGEREF _Toc249342536 \h  23

  HYPERLINK \l "_Toc249342537"  A.  Determination of Eligibility
PAGEREF _Toc249342537 \h  23

  HYPERLINK \l "_Toc249342538"  B.  Regulatory Decision	  PAGEREF
_Toc249342538 \h  24

  HYPERLINK \l "_Toc249342539"  C.  Labeling	  PAGEREF _Toc249342539 \h
24

  HYPERLINK \l "_Toc249342540"  VII.   ACTIONS REQUIRED BY THE APPLICANT
  PAGEREF _Toc249342540 \h  24

  HYPERLINK \l "_Toc249342541"  A.  Reporting of Adverse Effects and
Hypersensitivity Incidents	  PAGEREF _Toc249342541 \h  25

  HYPERLINK \l "_Toc249342542"  VIII.  GLOSSARY OF ACRONYMS AND
ABBREVIATIONS	  PAGEREF _Toc249342542 \h  26

  HYPERLINK \l "_Toc249342543"  IX.  BIBLIOGRAPHY	  PAGEREF
_Toc249342543 \h  27

  HYPERLINK \l "_Toc249342544"  A. Studies Submitted in Support of
Trichoderma asperellum strain ICC 012	  PAGEREF _Toc249342544 \h  27

  HYPERLINK \l "_Toc249342545"  B.  Environmental Protection Agency Risk
Assessment Memorandums	  PAGEREF _Toc249342545 \h  30

  HYPERLINK \l "_Toc249342546"  C. Other References Cited	  PAGEREF
_Toc249342546 \h  30

  HYPERLINK \l "_Toc249342547"  APPENDIX A – MICROBIAL PESTICIDE DATA
REQUIREMENTS	  PAGEREF _Toc249342547 \h  32

  HYPERLINK \l "_Toc249342548"  APPENDIX B – Trichoderma asperellum
strain ICC 012  Products	  PAGEREF _Toc249342548 \h  37

BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Science Reviews

Ibrahim Barsoum, Ph.D.				   Product Analysis, Human Health

John Kough, Ph.D.					   Product Analysis, Human Health

Shannon Borges					   Environmental Effects

Zigfridas Vaituzis, Ph.D.
Environmental Effects

Regulations

Sheryl Reilly, Ph.D.				               Chief, Microbial Pesticides
Branch

Susanne Cerrelli                     				   Regulatory Action Leader

I.  EXECUTIVE SUMMARY

The Biopesticides and Pollution Prevention Division (BPPD) received
applications to register, under Section 3(c)(5) of the Federal
Insecticide, Fungicide and Rodenticide Act (FIFRA), two products
containing the active ingredient, Trichoderma asperellum strain ICC 012,
as a microbial fungicide.  The manufacturing-use product (MP;
Trichoderma asperellum strain ICC 012 Technical, EPA  File Symbol
80289-RR) is intended to be used to formulate end-use pesticide products
with Trichoderma asperellum strain ICC 012.  The end-use product (EP;
Tenet WP, Bioten WP, Remedier WP; Tenet  T&O; EPA File Symbol 80289-O)
contains 2% Trichoderma asperellum strain ICC 012, as well as 2% of
another similar microbial active ingredient, Trichoderma gamsii strain
ICC 080.  The EP is intended to control pathogenic fungi on several food
and non-food crops, including ornamentals, fruiting vegetables, leafy
vegetables, cole crops, legumes, aromatic herbs, cucurbits, berries and
small fruits, and turf.  Depending on the use, the EP may be applied as
a cutting and bare root application, substrate mix, greenhouse and
nursery drench, broadcast application, or through chemigation.

The active ingredient, Trichoderma asperellum strain ICC 012 is a strain
of asexual fungi that was originally classified as Trichoderma harzianum
and subsequently recharacterized.  Trichoderma asexual fungi are
isolated primarily from soil and decomposing matter.

Trichoderma asperellum strain ICC 012 was isolated from a soil in
central Italy that was found to suppress plant disease.  Trichoderma
asperellum strain ICC 012 is used for control of many soil borne fungal
plant pathogens (i.e., Pythium species (spp.), Phytophthora spp.,
Sclerotinia spp., Sclerotium spp., Thielaviopsis basicola, Rhizoctonia
spp., Verticillum spp.)    Trichoderma asperellum strain ICC 012 acts as
a pathogen antagonist, colonizing in soil and around plant roots to
compete with plant pathogenic fungi for living space and nutrients.
Moreover, Trichoderma asperellum strain ICC 012 also uses enzymes to
attack the cell walls of pathogenic fungi.

Adequate mammalian toxicology data, data waivers, and other information
were submitted to fulfill the Tier I acute toxicity data requirements
for the registration of Trichoderma asperellum strain ICC 012 as a
microbial fungicide.

The toxicological data demonstrated that Trichoderma asperellum strain
ICC 012 is not toxic, infective or pathogenic to mammals.  No acute,
sub-chronic, chronic, immune, endocrine, or non-dietary exposure issues
were identified. The intended uses of this microbial fungicide do not
pose a dietary risk to the U.S. population in general, including infants
and children.  Dietary exposure via drinking water is also not expected
to pose harm to populations, because the microbial fungicide is not
known to grow or thrive in aquatic environments, nor would be expected
to survive municipal treatment of drinking water. Further, in the
unlikely event that humans are exposed to Trichoderma asperellum strain
ICC 012 from drinking water, the toxicological data base indicates no
adverse effects would be expected.

The potential for aggregate, non-occupational exposure from agricultural
applications is unlikely, because use sites identified for the subject
active ingredient are not expected to be in close proximity to
residential areas.  However, the product is also intended to be used in
a residential setting, and if inadvertent residential exposures occur
from agricultural applications, the low toxicity of the active
ingredient would not be expected to cause adverse effects to humans.

A final rule establishing an exemption from the requirement of a
tolerance was signed (publication date will be entered here), and
published in the Federal Register (publication citation will be entered
here).

Exposure of non-target organisms to Trichoderma asperellum strain ICC
012 is possible from the intended uses as a microbial fungicide.
Isagro, S.p.A. submitted studies and data waiver rationales to satisfy
data requirements for non-target organism risk assessment with the
active ingredient (TGAI).  Although not a data requirement for microbial
fungicides, the applicant submitted one acceptable non-target insect
study conducted on the predatory mite (Typhlodromus pyri).  However,
since Trichoderma harzianum has shown adverse effects in a coleopteran
insect (bark beetles), the following precautionary labeling statement
must be included on all end-products containing Trichoderma asperellum
strain ICC 012:  “This product may pose a hazard to beneficial
coleopteran species. Do not apply this product within ¼ mile of
endangered beetle habitat." In order to remove this precautionary label
language, acceptable coleopteran data (for example using the ladybird
beetle) must be submitted that demonstrate that Trichoderma asperellum
strain ICC 012 does not pose a hazard to these non-target insects.

The information and published reports provided by the applicant are
sufficient to satisfy the Tier I non-target organism data requirements
for the active ingredient, Trichoderma asperellum strain ICC 012,
contained in their MP and the EP.  Further testing of non-target
organisms at higher tier levels is not required.

The study conducted to test the toxicity and pathogenicity to honey bees
(Apis mellifera) was unacceptable, due to the short duration (48 hours)
of exposure indicated in the study submitted.  Honeybee studies should
be long enough (e.g., 30 days) to allow for the observation of latent
pathogenic effects.  Because of concerns for potential effects to
honeybees, the statement: “Do not apply this product while bees are
actively visiting the treatment area” is included on the end product
label, and must be included on the labels of any future products
containing this microbial active ingredient, unless acceptable data are
submitted that demonstrate its low toxicity and pathogenicity to
honeybees.

Consistent with the new policy of making registration actions more
transparent, Trichoderma asperellum strain ICC 012 is subject to a 30
day comment period as a “new active ingredient” whose registration
would result in a “first outdoor use” and a “first residential
use.”  While a final decision on registration is contingent upon
review and consideration of public comments, EPA presently believes that
based on the risk assessment and information submitted in support of the
registration of Trichoderma asperellum  strain ICC 012  that it is in
the best interests of the public and the environment to issue the
registration for Trichoderma asperellum strain ICC 012. The basis for
this preliminary decision can be found in the risk assessment for
Trichoderma asperellum strain ICC 012, which is characterized in this
BRAD.  As discussed above, acute toxicity data for demonstrate that it
has low toxicity (category III and IV).  EPA has no concerns for
non-target organisms, included threatened or endangered species, exposed
to Trichoderma asperellum strain ICC 012 when it is used in accordance
with approved label directions.  EPA has not identified any toxic
endpoints for non-target mammals, birds, plants, aquatic, or soil
organisms. Thus, EPA concludes that use of the microbial fungicide
Trichoderma asperellum strain ICC 012 in accordance with the proposed
label directions will not cause any unreasonable adverse effects on the
environment.

The Environmental Protection Agency (EPA) considered information
submitted in support of an application for a registration under section
Section 3(c)(5) of the Federal Insecticide, Fungicide and Rodenticide
Act (FIFRA) for the microbial fungicide Trichoderma asperellum strain
ICC 012 and determined that these data and information adequately
satisfy current data requirements (refer to 40 CFR Subpart U §
158.2000).  If the Agency receives comments during the 30 day public
comment period that inform EPA’s initial decision, EPA will address
such new information and take appropriate action.

II.  ACTIVE INGREDIENT OVERVIEW

	Biological Name:		Trichoderma asperellum strain ICC 012

Culture Deposit:	CABI Bioscience International Mycological Institute-
Egham, UK under IMI CC No. 392716

	Trade/Other Names:  	Bioten WP; Tenet WP; Remedier WP; Tenet T&O

Office of Pesticide

Programs (OPP)

Chemical Code:		119208

	Type of Pesticide:		Microbial Pesticide, fungicide

					See   HYPERLINK  \l "Table1_AppB"  Appendix B  for specific
information (i.e.,

use sites, application rate, method of application,

formulation type, and target pests)

III.  REGULATORY BACKGROUND

On February 8, 2008, Mel Graben  of Isagro, USA (address: 430 Davis
Drive, Suite 240, Morrisville, NC 27560), acting as the United States
authorized agent for Isagro S.p.A.,  (address: Centro Uffici San-Edifico
D-ala 3, Via Caldera, 21-20153 Milan, Italy) submitted an application to
register Bioten WP (EPA File Symbol 80289-O)  and  Trichoderma
asperellum strain ICC 012 (EPA File Symbol 80289- RR) under Section 3 of
the Federal Insecticide, Fungicide, and Rodenticide Act. On October 29,
2008, the EPA announced receipt of this application to register a
pesticide product containing a new active ingredient [ HYPERLINK
"http://www.epa.gov/fedrgstr/EPA-PEST/2008/March/Day-28/p6266.htm" 73
Federal Register (FR)  64325] and opened a 60-day public comment period
on the receipt of this application.

In response to this notice of receipt, EPA did not receive any
substantive comments relevant to the subject Trichoderma pesticide
products.

Concurrent with their registration application, and under the Federal
Food, Drug, and Cosmetic Act (FFDCA), as amended by the Food Quality
Protection Act of 1996, Isagro S.p.A. submitted a petition to establish
an exemption from the requirement of a tolerance for Trichoderma
asperellum  strain ICC 012 [Pesticide Petition (PP) 8F 7326]. In the
Federal Register dated November 12, 2008 (73 FR  66897), the EPA
announced that Isagro, S.p.A., proposed to establish an exemption from
the requirement of a tolerance for residues of the microbial pesticide,
Trichoderma asperellum strain ICC 012.   One comment from an anonymous
American citizen was received following the publication of a notice of
filing of a petition under the FFDCA.

EPA is proposing to promulgate a final rule establishing the exemption
from tolerance for Trichoderma asperellum strain ICC 012.  EPA has
placed the draft final rule that would establish such a tolerance
exemption in the docket along with this proposed FIFRA decision to
permit comment on both actions the Agency is considering

IV.  RISK ASSESSMENT

On October 26, 2007, the Agency issued a Final Rule in the Federal
Register on the data requirements to support registration of biochemical
and microbial pesticides, and updated the definitions for biochemical
and microbial pesticides (  HYPERLINK
"http://www.epa.gov/fedrgstr/EPA-TOX/2007/October/Day-26/t20828.htm"  72
FR 61002 ). The rule became effective on December 26, 2007. The data and
information evaluated for this Biopesticides Registration Action
Document (BRAD) were considered in light of these requirements.

The classifications that are found for each data submission are assigned
by EPA science reviewers and are an indication of the usefulness of the
information contained in the documents for risk assessment. A rating of
“ACCEPTABLE” indicates the study is scientifically sound and is
useful for risk assessment. A “SUPPLEMENTAL” rating indicates the
data provide some information that can be useful for risk assessment.
The studies may have certain aspects determined not to be scientifically
acceptable (“SUPPLEMENTAL: UPGRADABLE”). If a study is rated as
“SUPPLEMENTAL: UPGRADABLE,” the Environmental Protection Agency
always provides an indication of what is lacking or what can be provided
to change the rating to “ACCEPTABLE.” If there is simply a
“SUPPLEMENTAL” rating, the reviewer will often state that the study
is not required by the current 40 CFR Part 158. Both “ACCEPTABLE”
and “SUPPLEMENTAL” studies may be used in the risk assessment
process as appropriate. An “UNACCEPTABLE” rating indicates that new
data need to be submitted.

For product-specific acute toxicity data requirements, toxicity
categories are assigned based on the hazard(s) identified from studies
and/or information submitted to the Agency. The product is classified
into Toxicity Category I, II, III, or IV, where Toxicity Category I
indicates the highest toxicity and Toxicity Category IV indicates the
lowest toxicity.

A.  Product Analysis Assessment

Bioten™ WP, containing Trichoderma asperellum strain ICC 012 as an
active ingredient, have been satisfied by either acceptable guideline
studies or waiver rationales. For a comprehensive guideline-by-guideline
summary of the product analysis data requirements described in sections
IV(A)(1), IV(A)(2), and IV(A)(3), refer to   HYPERLINK  \l "Table1"
Table 1  in Appendix A.

	1.  Product Chemistry and Composition

Trichoderma asperellum strain ICC 012 Technical is an MPCA soil
fungicide for formulating use only.  The product, contains 99.9% w/w
Trichoderma asperellum strain ICC 012 as active ingredient with a
minimum and nominal content of 1 x 109 and 2.5 x 109 conida cfu/g dry
weight, respectively.  The label indicates that the product contains
Trichoderma asperellum strain ICC 012 and 0.10% inert ingredients.  The
strain ICC 012, originally characterized as Trichoderma harzianum, has
been reclassified as Trichoderma asperellum and also has been
characterized by molecular biology techniques.  Trichoderma asperellum
strain ICC 012 was isolated in 1987 by the laboratories of the “Centro
Esperienze e Ricerche – SIAPA Company” from a soil of central Italy
that suppressed plant disease.  The strain has been deposited in the
CABI Bioscience International Mycological Institute-Egham, UK IMI CC No.
392716.

No relationships are known between Trichoderma genus and any pathogen of
mammals or plants.  The extensive Moniliaceae family which includes
phytopathogenic genera Botrytis and Verticillium and human pathogen
Blastomyces; includes Trichoderma, but Trichoderma  and closely related
genera are not known to be mammalian pathogens.

The submitted data satisfied the requirements for manufacturing process
and discussion of formation of unintentional ingredients. [(OPPTS)
Guidelines 885.1100, 885.1200, and 885.1300]

	2.  Analysis and Certified Limits

Results of a 5-batch preliminary analysis were provided and the
requirement for analysis of samples has been satisfied.  The requirement
for certified limits has been satisfied.

3.  Physical and Chemical Characteristics

The submitted data and waiver rationales satisfied the requirements for
the physical and chemical characteristics, including color, physical
state, odor, stability to normal and elevated temperatures, metals, and
metal ions, storage stability, miscibility, corrosion characteristics,
pH, viscosity, and density/relative density/bulk density (specific
gravity).

B.  Human Health Assessment

	1.  Toxicology

Bioten™ WP, which contains Trichoderma asperellum strain ICC 012. Tier
II and Tier III studies were not required for Trichoderma asperellum
strain ICC 012 based on the lack of acute toxicity/pathogenicity in the
Tier I studies.

For a comprehensive guideline-by-guideline summary of the toxicology
data requirements and additional studies described in sections
IV(B)(1)(a), IV(B)(1)(b), and IV(B)(1)(c), refer to   HYPERLINK  \l
"Table2"  Table 2  in Appendix A.

a.  Acute Toxicity/Pathogenicity – Tier I (  HYPERLINK
"http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&sid=76805ba87ba7a2
c69b53feed9af452bb&rgn=div8&view=text&node=40:<IP_ADDRESS>.<IP_ADDRESS>&idno=40"
 40 CFR § 158.2140 )

Acute Oral Toxicity – Rat [OPPTS Guideline 885. 3550; (MRID# 47345901)

In an acute oral toxicity study groups of fasted, 6-7 week old rats
(5/sex) were given a single oral dose of Trichoderma asperellum strain
ICC 012 (Trichoderma asperellum  conidia 4.2 x 109 CFU/g) in 0.9% NaCl
solution at a dose of 2000 mg/kg bw in a limit test.  The animals were
then observed for a period of 14 days. The following results of Oral
LD50 were: Males > 2000 mg/kg bw, Females > 2000 mg/kg bw, Combined >
2000 mg/kg bw.  No mortality occurred during the study.  Based on the
results of this study, Trichoderma asperellum strain ICC 012 was not
toxic at 2000 mg/kg bw.  Microbial enumeration was not performed;
therefore, the infectivity and pathogenicity of Trichoderma asperellum
are unknown. There were no treatment related clinical signs, necropsy
findings, or changes in body weight. This study was rated SUPPLEMENTAL
but was upgraded to “ACCEPTABLE” by clearance of the organism
observed in the pulmonary toxicity/pathogenicity study.

Acute Pulmonary Toxicity/Pathogenicity Studies– Rats (OPPTS Guideline
885.3150;

MRID# 47345903, 47345904).  Groups of fasted, 44-55 days old rats
(31/sex) were exposed by the intratracheal route to Trichoderma
asperellum strain ICC 012 (Trichoderma asperellum conidia 4.2 x 109
CFU/g) in a 0.1% solution of Tween 20 in water for injection at a dose
of 1 x 107 CFU/animal.  Animals were then observed for up to 22 days.
Rats in the control group were administered the vehicle only.  Rats in
the reference groups were administered inactivated test item. Samples of
feces, lungs, lymph nodes, kidneys, brain, liver, spleen, and blood were
taken for the determination of microbial enumeration.  The viable count
was 4.2 x 109 CFU/g and the greatest density was detected in lung
tissue. Pulmonary LD50 Males > 1 x 107 CFU/animal, Females > 1 x 107
CFU/animal, Combined > 1 x 107 CFU/animal. No mortality occurred. Based
on these results, Trichoderma asperellum  strain ICC 012 is of LOW
Toxicity and is not infective or pathogenic in the rat. This study was
rated “ACCEPTABLE” for risk assessment purposes.

Acute Intraperitoneal Injection Toxicity– Rat (OPPTS Guideline
885.3200; (MRID# 47345902).  In an acute intraperitoneal injection
toxicity and pathogenicity study groups of fasted, 6-7 week old rats
(3/sex) were injected with Trichoderma asperellum strain ICC 012
(Trichoderma asperellum conidia 4.2 x 109 CFU/g) in 0.9% NaCl solution
at a dose of 1 x 108 CFU/g in a limit test.  Animals were then observed
for up to 21 days.  Control animals were injected with 0.9% NaCl
solution only.  Trichoderma asperellum strain ICC 012 is NOT TOXIC based
on these results.  Since microbial enumeration was not performed, the
infectivity was uncertain. There were no treatment-related clinical
signs, necropsy findings, or changes in body weight.  This study was
rated “SUPPLEMENTAL,” but was upgraded to “ACCEPTABLE” based on
the clearance observed in the pulmonary toxicity/pathogenicity study
described above.

Acute Oral Toxicity/Pathogenicity– Rats   (OPPTS Guideline 870.1100;
MRID# 47346301).  In an acute oral toxicity study (MRID 47346201),
groups of fasted, 6-7 week old CD/CRL:CD rats (5/sex) were given a
single oral dose of Bioten WP (originally referred to as Remedier WP in
the acute studies below)(Trichoderma conidia (1.2 x 108 CFU/g);
Trichoderma asperellum strain ICC 012 (originally classified as T.
harzianum)  (7.8 x 107 CFU/g); Trichoderma gamsii  strain ICC 080 (4.2 x
107 CFU/g)) in 0.9% NaCl solution at a dose of 2000 mg/kg bw in a limit
test.  The animals were then observed for a period of up to 14 days.
Oral LD50 were as follows: Males > 2000 mg/kg bw, Females > 2000 mg/kg
bw , Combined > 2000 mg/kg bw. Limit test: no mortality occurred during
the study. Based on the results of this study BiotenWP (Trichoderma
asperellum 7.8 x 107 CFU/g; Trichoderma gamsii  4.2 x 107 CFU/g) is not
toxic and has an LD50 greater than 2000 mg/kg bw.(EPA Toxicity Category
III.) Microbial enumeration was not performed, therefore, the
infectivity and pathogenicity of Trichoderma conidia, Trichoderma
asperellum strain ICC 012, and Trichoderma gamsii strain ICC 080, are
unknown. There were no treatment related clinical signs, necropsy
findings or changes in body weight. This study was rated
“ACCEPTABLE” for risk assessment purposes.

Acute Dermal Toxicity (OPPTS Guideline 870.1200; MRID# 47346302)

In an acute dermal toxicity study (MRID 43746302), groups of fasted, 6-7
week old CD/CRL:CD rats (5/sex) were given a single dermal dose of
BiotenWP (Trichoderma conidia (1.2 x 108 CFU/g); Trichoderma asperellum
strain ICC 012 (7.8 x 107 CFU/g); Trichoderma gamsii strain ICC 080 (4.2
x 107 CFU/g)) in 0.9% NaCl at a dose of 2000 mg/kg bw for 24 hours to an
area of approximately 5 x 6 cm (~10% of body surface).  Following
exposure, the animals were observed for a period of 14 days. Dermal LD50
were as follows: Males > 2000 mg/kg bw,Females > 2000 mg/kg bw, Combined
> 2000 mg/kg bw. Limit test; no mortality occurred during the study.
Based on the results of this study Remedier WP (Trichoderma asperellum
7.8 x 107 CFU/g; Trichoderma gamsii  4.2 x 107 CFU/g) is of MODERATE
Toxicity, EPA Toxicity Category III. This study was rated
“ACCEPTABLE” for risk assessment purposes.

Acute Inhalation Toxicity (OPPTS Guideline 870.1300; MRID# 47346303)

In an acute inhalation toxicity study (MRID 47346303), groups of fasted,
44-55 day old CD/CRL:CD rats (5/sex) were exposed by the inhalation
route to Bioten WP (Trichoderma conidia (1.2 x 108 CFU/g); Trichoderma
asperellum strain ICC 012 (7.8 x 107 CFU/g); Trichoderma gamsii strain
ICC 080 (4.2 x 107 CFU/g)) as supplied at a concentration of 5.20 mg/L.
Animals then were observed for 14 days. The MMAD was 2.573 µm and the
GDS was 11.375. Inhalation LC50were as follows: Males > 5.20 mg/L,

Females > 5.20 mg/ L, Combined > 5.20 mg/L. No mortality occurred during
the study. Bioten WP (Trichoderma asperellum 7.8 x 107 CFU/g;
Trichoderma gamsii 4.2 x 107 CFU/g) is of LOW Toxicity, EPA Toxicity
Category IV, based on lack of mortality in male and female rats. There
were no treatment related clinical signs, necropsy findings or changes
in body weight. This study was rated “ACCEPTABLE” for risk
assessment purposes. Toxicity Category IV.

Acute Eye Irritation – Rabbit (OPPTS Guideline 870.2400; MRID#
473463034)

In a primary eye irritation study (MRID 47346304), 100 mg of Bioten WP
(Trichoderma conidia (1.2 x 108 CFU/g); Trichoderma asperellum strain
ICC 012 (7.8 x 107 CFU/g); Trichoderma gamsii strain ICC 080 (4.2 x 107
CFU/g)), as supplied, was instilled into the right conjunctival sac of 3
male fasted young adult Himalayan rabbits  for 24 hours. Animals then
were observed for 72 hours. Irritation was scored by the method that was
not named but similar to the method of Draize (1959). Conjunctival
redness was observed in all animals 1 hour after instillation, and
resolved by 24 hours.  The cornea and the iris were not affected by
instillation of the test item. In this study, Bioten WP (Trichoderma
asperellum 7.8 x 107 CFU/g; Trichoderma gamsii 4.2 x 107 CFU/g) was not
an eye irritant and is in EPA Toxicity Category IV. This study was rated
“ACCEPTABLE” for risk assessment purposes.

Acute Dermal Irritation Toxicity- Rabbit (OPPTS 870.2500; MRID #
47346305)

In a primary dermal irritation study (MRID 47346305), fasted young adult
Himalayan rabbits (3 males) were dermally exposed to 500 mg of Bioten WP
(Trichoderma conidia (1.2 x 108 CFU/g); Trichoderma asperellum strain
ICC 012 (7.8 x 107 CFU/g); Trichoderma gamsii strain ICC 080 (4.2 x 107
CFU/g)) in aqua ad iniectabilia (water for injection) for 4 hours on a
shaved area of 6 cm2.  Animals then were observed for 6 days.
Irritation was scored by the method that was not named but similar to
the method of Draize (1944). Very slight erythema was observed on all
three animals 24 hours through 5 days after patch removal.  Very slight
edema was observed on one animal 72 hours througho 4 days after patch
removal.  The Primary Irritation Index (PII) was 0.8. In this study,
Remedier WP (Trichoderma asperellum 7.8 x 107 CFU/g; Trichoderma gamsii
4.2 x 107 CFU/g) is slightly irritating and is in EPA Toxicity Category
IV. This study was rated “ACCEPTABLE” for risk assessment purposes.

Skin Sensitization Study-Guinea Pigs	OPPTS 870.2600; MRID # 47346306)

   SEQ CHAPTER \h \r 1 In a skin sensitization study (MRID 47346306)
with Bioten WP (Trichoderma conidia (1.2 x 108 CFU/g); Trichoderma
asperellum strain ICC 012 (7.8 x 107 CFU/g); Trichoderma gamsii strain
ICC 080 (4.2 x 107 CFU/g)) in 0.9% NaCl, young adult Dunkin-Hartley
guinea pigs (15 males) were tested using the method of Magnusson and
Kligman (Maximization Test). The test substance was administered as a
suspension for intradermal application and applied as a suspension for
topical application.  Benzocaine was used as a positive control.
Discrete or patchy erythema was observed on all test animals exposed to
the 10% suspension of Bioten WP (Trichoderma asperellum  and Trichoderma
gamsii  wettable powder) in 0.9% NaCl solution 25 and 48 hours after the
induction phase.  Some animals exposed to a 50% suspension of Bioten WP
(Trichoderma asperellum and Trichoderma gamsii wettable powder) in 0.9%
NaCl solution had moderate, confluent erythema 49 and 72 hours after
application.  There were no skin reactions in the test animals 24, 48,
or 72 hours after the final challenge using a 1% suspension of BiotenWP
(Trichoderma asperellum and Trichoderma gamsii  wettable powder) in 0.9%
NaCl solution.  In this study, (Trichoderma asperellum 7.8 x 107 CFU/g;
Trichoderma gamsii 4.2 x 107 CFU/g) was not a dermal sensitizer. This
study was rated “ACCEPTABLE” for risk assessment purposes.

	b.	Acute Toxicology and Subchronic Toxicity/Pathogenicity – Tier II;

	    	Reproductive Fertility Effects, Carcinogenicity, Immunotoxicity,
and

	   	Infectivity/Pathogenicity Analysis – Tier III (  HYPERLINK
"http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&sid=9061faa75a746f
eda99a9bbe8072f5d3&rgn=div8&view=text&node=40:<IP_ADDRESS>.<IP_ADDRESS>&idno=40"
 40 CFR § 158.2140 )

Tier II and Tier III studies were not required for Trichoderma
asperellum strain

ICC 012 based on the lack of acute toxicity/pathogenicity in the Tier I
studies.

	c.  Effects on the Endocrine System

Section 408(p) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
requires EPA to develop a screening program to determine whether certain
substances (including all pesticide active and other ingredients) “may
have an effect in humans that is similar to an effect produced by a
naturally occurring estrogen, or such other endocrine effect as the
Administrator may designate.” Following the recommendations of its
Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC),
EPA determined that there were scientific bases for including, as part
of its program, androgen and thyroid hormone systems, in addition to the
estrogen hormone system. The Environmental Protection Agency also
adopted EDSTAC’s recommendation that the Program include evaluations
of potential effects on wildlife.

The Agency has no information to suggest that Trichoderma asperellum
strain

ICC 012 has an effect on the endocrine system.  The submitted acute
pulmonary toxicity/pathogenicity study in rodents indicated that
following pulmonary exposure, the immune system is still intact and able
to process and clear the active ingredient.  Trichoderma asperellum
strain ICC 012 is a ubiquitous organism in the environment and there
have been no reports of the organism affecting endocrine systems.
Therefore, it is unlikely that this organism would have estrogenic or
endocrine effects and it is practically non-toxic to mammals.

Additional data, specifically on the endocrine effects of this microbial
pesticide, are not required at this time. When the appropriate screening
and/or testing protocols being considered under the Agency’s Endocrine
Disruptor Screening Program (EDSP) have been developed and vetted,
Trichoderma asperellum strain ICC 012 may be subjected to additional
screening and/or testing to better characterize effects related to
endocrine disruption.

	2.  Dietary Exposure and Risk Characterization

Dietary exposure to the microbial pesticide is likely to occur, but the
lack of acute oral toxicity, infectivity, and pathogenicity support the
establishment of an exemption from the requirement of a tolerance for
Trichoderma asperellum strain ICC 012.

Dietary exposure to the microbial active ingredient is expected to be
minimal.  The product is typically applied to soil but sometimes may be
applied when the crops are growing in the field, resulting in residues
on the crops.  The Agency expects residues on food to be minimal because
this pesticide is typically applied to soil, rather than crops.
Moreover, Trichoderma lives in soils and is unlikely to live on the
plants because any spores that do end up on the plant due to application
would decrease over time due to weathering, desiccation and ultraviolet
radiation which can kill even quiescent forms of the fungus.  In the
unlikely event that the applied fungus grew on edible portions of a
treated crop, there is no hazard expected from these residues, as
demonstrated by the results of testing which show no toxicity or
pathogenicity in treated animals when dosed with the fungus at orders of
magnitude above any expected exposure to the microbial pesticide.  (See
 HYPERLINK  \l "Acute_Toxicity_Pathogenicity_TierI"  section IV(B)(1)(a)
.)

3.   Drinking Water Exposure and Risk Characterization

Drinking water exposure is expected to be negligible because the
microbial fungicide will not be applied to water.   Further Trichoderma
asperellum is a soil microorganism, and would not proliferate in aquatic
environments.   Moreover, the Agency believes that Trichoderma within
the soil will not likely percolate into water because of the large size
of the fungal spores and the fact that they adhere to soil particles.
Even in the unlikely event that dietary exposure occurs through drinking
water, the Agency concludes that there is a reasonable certainty that no
harm will result because of the lack of acute oral
toxicity/pathogenicity to mammals as previously described.

	4.  Acute and Chronic Dietary Exposure and Risks for Sensitive

 	     Subpopulations, Particularly Infants and Children

Section 408(b)(2)(C) of the FFDCA provides that EPA shall assess the
available information about consumption patterns among infants and
children, special susceptibility of infants and children to pesticide
chemical residues, and the cumulative effects on infants and children of
the residues and other substances with a common mechanism of toxicity.
In addition, FFDCA section 408(b)(2)(C) also provides that EPA shall
apply an additional tenfold margin of safety for infants and children in
the case of threshold effects to account for prenatal and postnatal
toxicity and the completeness of the database unless EPA determines that
a different margin of safety will be safe for infants and children.
Margins of exposure (safety), which are often referred to as uncertainty
factors, are incorporated into EPA risk assessments either directly or
through the use of a margin of exposure analysis, or by using the
uncertainty factors in the calculation of dose levels that pose no
appreciable risk.

Based on the acute toxicity and pathogenicity data discussed in
HYPERLINK  \l "Acute_Toxicity_Pathogenicity_TierI"  section IV(B)(1)(a)
, the Environmental Protection Agency concludes that there is a
reasonable certainty that no harm to sensitive subpopulations, including
infants, children, and adults, will result from the use of Trichoderma
asperellum strain ICC 012. This includes all anticipated dietary
exposures and all other exposures for which there is reliable
information. The Agency has arrived at this conclusion because the data
available on Trichoderma asperellum strain

ICC 012 does not demonstrate toxic, pathogenic, or infective potential
to mammals. Thus, there are no threshold effects of concern and, as a
result, the provision requiring an additional margin of safety does not
apply.  Further, the considerations of consumption patterns, special
susceptibility, and cumulative effects do not apply to pesticides
without a demonstrated significant adverse effect.

	5.  Occupational, Residential, School, and Daycare Exposure and Risk

	     Characterization

Occupational Exposure and Risk Characterization

In light of the Tier I acute toxicity/pathogenicity studies, which did
not show any toxic via oral, pulmonary, or intraperitoneal injection
routes of exposure (  HYPERLINK  \l "Acute_Toxicity_Pathogenicity_TierI"
 see section IV(B)(1)(a) ), or pathogenic effects to rats via pulmonary
exposure, handler exposure to Trichoderma asperellum strain ICC 012 is
not expected to pose any undue risk. Regardless, requirements for the
use of appropriate personal protective equipment, and precautionary
statements are required on the product label to mitigate any potential
risks to pesticide handlers due to prolonged exposure. Handlers working
with Trichoderma asperellum strain ICC 012 must wear a long-sleeved
shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist
filtering respirator meeting NIOSH standards of at least N-95, R-95, or
P-95 when mixing, loading, or applying the product.

Residential, School, and Daycare Exposure and Risk Characterization

Trichoderma asperellum strain ICC 012 is a naturally occurring microbe
and is ubiquitous in the environment.  Trichoderma asperellum strain ICC
012 will be applied to substrate mixes, ornamental plants, agricultural
fields, turf, and various plants grown in greenhouses. Although some
applications to turf  or ornamental plants may be in residential areas,
non-dietary exposure would be expected to be below the Agency’s level
of concern because of its low toxicity classification, and because the
lab results indicate Trichoderma asperellum strain ICC 012  is not
pathogenic to mammals.

	6.  Aggregate Exposure from Multiple Routes Including Dermal, Oral, and

                 Inhalation

In examining aggregate exposure, Section 408 of the FFDCA directs EPA to
consider available information concerning exposures from the pesticide
residue in food and all other non-occupational exposures, including
drinking water from ground water or surface water and exposure through
pesticide use in gardens, lawns, or buildings (residential and other
indoor uses).

Trichoderma asperellum strain ICC 012 is a naturally occurring microbe
and is ubiquitous in the environment.  Trichoderma asperellum strain ICC
012 will be applied to substrate mixes, ornamental plants, agricultural
fields, turf, and various plants grown in greenhouses. Although some
applications to turf  or ornamental plants may be in residential areas,
non-dietary exposure would be expected to be below the Agency’s level
of concern because of its low toxicity classification, and because the
lab results indicate Trichoderma asperellum strain ICC 012  is not
pathogenic to mammals.

7.  Cumulative Effects

Section 408(b)(2)(D)(v) of the FFDCA requires the Agency to consider the
cumulative effect of exposure to Trichoderma asperellum strain ICC 012
and to other substances that have a common mechanism of toxicity. These
considerations include the possible cumulative effects of such residues
on infants and children.  Based on tests in mammalian systems,
Trichoderma asperellum strain ICC 012 does not appear to be toxic to
humans via dietary and pulmonary exposure.  Therefore, the requirement
to consider cumulative effects does not apply.

	8.  Risk Characterization

The Agency considered human exposure to Trichoderma asperellum strain
ICC 012 in light of the standard for registration and safety factors in
FIFRA and FFDCA, as amended by the FQPA. A determination has been made
that no unreasonable adverse effects to the United States population in
general, including infants and, will result from the use of Trichoderma
asperellum strain ICC 012 when used in accordance with EPA-approved
labeling.

C.  Environmental Assessment

Exposure of non-target organisms to Trichoderma asperellum strain ICC
012 is a possible result of the proposed uses of the EP containing this
active ingredient.  Isagro, S.p.A. submitted studies and data waiver
rationale to satisfy data requirements for non-target organism risk
assessment with the active ingredient (TGAI).  The following is a review
of the information submitted to support registration and an assessment
of environmental risk based on these data.

		1.  Summary of Non-target Organism Testing and Waiver Rationales

The data and other information provided to the Agency in support of
registrations for a manufacturing product and end use microbial
pesticide product is sufficient to satisfy the Tier I non-target
organism data requirements for the active ingredient, Trichoderma
asperellum strain 012.  Table 3 in Appendix A provides the status of the
data requirements for the TGAI.  Summaries of the studies and data
waiver rationales that were submitted are discussed at the end of this
section.

a.  Avian Oral Toxicity/Pathogenicity (OPPTS Guideline 885.4050); Avian
Inhalation Toxicity/Pathogenicity (OPPTS Guideline 885.4100)

The applicants submitted waivers for the required avian
toxicity/pathogenicity testing.  Justification for these waivers was
based upon the temperatures at which Trichoderma species can grow
compared with avian body temperatures. The rationale cited data that
showed that Trichoderma species grow at temperatures below 37°C,
whereas normal bird body temperatures are typically above 38°C, with
active body temperatures above 40°C (Prinzinger et al. 1991).  As the
applicant states, EPA has acknowledged the validity of this rationale in
the past (USEPA 2008).  The applicant also argued that no toxic or
pathogenic effects to birds should be expected, since none were observed
in the rat acute oral toxicity/pathogenicity tests with the active
ingredient or the EP.  However, the comparability of the effects between
these two taxa for Trichoderma asperellum strain ICC 012 is not known.

Based upon the growth temperature requirements for Trichoderma
asperellum strain ICC 012 compared with the higher body temperature of
birds, adverse effects to birds are not expected due to exposure to
Trichoderma asperellum  strain ICC 012.  This rationale is sufficient to
fulfill the data requirement.

b.  Freshwater Fish Toxicity/Pathogenicity (OPPTS 885. 4200) and

(MRID No.: 47345905)

In a 30-day toxicity/pathogenicity study juvenile rainbow trout
(Oncorhynchus mykiss) received an aqueous exposure to nominal
concentrations of 6.25, 12.5, 25, 50, and 100 mg Trichoderma asperellum
strain ICC 012/L (corresponding to measured concentrations of 1.5 x 109,
3.0 x 109, 6.0 x 109, 1.2 x 1010, and 2.4 x 1010 cfu/L, respectively)
under semi-static conditions. A dilution water control and an attenuated
control were also included.  One fish died in each of the 6.25 mg/L and
100 mg/L test groups (the latter from jumping out of the aquarium),
which was not statistically significant from the controls. The 30-day
LC50 was empirically estimated to be greater than the highest nominal
concentration tested 100 mg/L (measured concentration 2.4 x 1010 cfu/L).
Pseudo-specific growth rate did not differ significantly from either
control in any test group except the 100 mg/L test group, in which the
fish gained significantly less weight.  Because of this, the NOAEC was
determined to be 50 mg/L (1.2 x 1010 cfu/L).  Excessive turbidity,
coloration, and bubbling in the test water was noted in the three
highest concentrations, and it is possible that the fouling of the water
by the test material contributed to the lower weight gain.  This study
was classified as “ACCEPTABLE.”

c.  Freshwater Invertebrate Toxicity/Pathogenicity (OPPTS 885.4240)

(MRID No.: 47345906 and 47346307)

In a 21-day toxicity/pathogenicity study, neonate Daphnia magna received
an aqueous exposure to nominal concentrations of 6.25, 12.5, 25, 50, and
100 mg Trichoderma asperellum strain ICC 012 /L (corresponding to
measured concentrations of 1.9 x 109, 1.9 x 109, 4.4 x 109, 7.6 x 109,
and 1.7 x 1010 cfu/L, respectively) under semi-static conditions.  A
dilution water control and an attenuated control containing sterile
filtrate were also included.  Survival was 95%, 95%, 75%, 90%, and 65%
in the 6.25, 12.5, 25, 50 and 100 mg/L test concentrations,
respectively.  Survival was 55% and 100% in the attenuated control and
negative control, respectively.  The Daphnia survival rate was
significantly reduced in the 100 mg/L and the sterile filtered test
media (attenuated control) when compared to the negative control;
however there was no delay of the first brood reproduction at any of the
test item concentrations and no significant toxic effect of the test
item at all nominal test concentrations on the mean reproduction rate
compared to the control.  The EC50 of the reproduction rate was
determined to be ≥100 mg/L (1.7 x1010 cfu/L), the 21-day NOEC for
Daphnia magna based on mortality was 50 mg/L (7.6 x 109).  The NOEC
(based on reproduction, dissemination) was ≥ 100 mg/L (1.7 x1010
cfu/L).  This study was classified as “ACCEPTABLE.”

A study was also submitted with the EP entitled, “Acute Toxicity of
Remedier WP to Daphnia magna in a 48-hour Immobilization Test” (MRID
473463-07).    This 48-hour Immobilization test (MRID No.: 47346307) was
performed with the end-use product.  In a 48-hour immobilization study,
Daphnia magna received an aqueous exposure to the nominal concentration
of 100 mg/L of Remedier WP under semi-static conditions (test and
control water was renewed at 24 hours).  The active ingredients in
Remedier WP are Trichoderma asperellum strain ICC 012 and Trichoderma
gamsii strain ICC 080 (corresponding to 3.2 x 108 cfu/L total, measured)
and are each contained in the product at 2% by weight.  A dilution water
control was also included.  No adverse effects were observed in daphnids
exposed to the 100 mg/L limit test concentration which indicates a
48-hour EC50 of >100 mg/L (3.2 x 108 cfu/L).  This study is classified
as unacceptable as a Tier I pathogenicity study for freshwater aquatic
invertebrate with a microbial pesticide due to the 48-hour duration.
The study was classified as “unacceptable”. It cannot be upgraded
and cannot be used to fulfill the pathogenicity data requirement for
either TGAI.

Non-target Insect Testing (OPPTS 885.4340) (MRID No.: 47346308)

This study is not a data requirement for microbial fungicides, but the
applicants submitted the study and it was reviewed by BPPD.  The
dose-response study was performed with the EP.  In a 14-day contact
toxicity/pathogenicity study, the predatory mite, Typhlodromus pyri, was
exposed to nominal concentrations of 0.062, 0.185, 0.556, 1.670, and 5.0
kg of Remedier WP/ha (corresponding to 0.002, 0.007, 0.022, 0.067, and
200 g total active ingredient/ha).  Mite mortality was monitored for the
first 7-days and the effects of Remedier WP on reproduction potential
were monitored in adult mites from day 7 thru 14.  The active
ingredients in Remedier WP are Trichoderma asperellum strain ICC 012 and
Trichoderma gamsii strain ICC 080 (corresponding to 1.2 x 108 cfu/L,
total).  A dilution water control and positive control were included in
the study.  Statistically significant mortality was not observed in
mites exposed to any of the Remedier WP treatment concentrations.  Thus
the LD50 was determined to be > 5 kg/ha, which is higher than the
highest broadcast application rate.  Confirmation of infection by the
Trichoderma active ingredients in dead mites was not performed.
Reproduction was affected in the 0.062 kg, 0.185 kg and 5.0 kg Remedier
WP/ha treatment groups, but no statistically significant effects on
reproduction occurred in the 0.556 kg or 1.670 kg Remedier WP/ha
treatment groups.  The study authors concluded that effects observed on
reproduction were not of biological significance and not due to exposure
to the test item.  However, these results are inconclusive and it
appears that the study design is not sensitive enough to detect
biologically significant reductions in reproduction in this species as a
result of exposure to the test material.  Because mortality is the
primary endpoint of concern, this study is classified as
“ACCEPTABLE.”

Honey Bee Testing (OPPTS 885.4380) (MRID No.: 47345907)

Adult honey bees (Apis mellifera L.) were exposed to Trichoderma
asperellum strain 012 technical in 48-hour contact and oral toxicity
tests.  Bees were exposed to 50 µg per bee Trichoderma asperellum
strain ICC 012 (2.1 x 105 cfu/bee) in the contact toxicity test and
111.5 µg/bee Trichoderma asperellum strain ICC 012 (4.7 x 105 cfu/bee)
in an oral toxicity test.  A negative control (sugar solution for the
oral test, water with 1% Adhasit [a spreader] for the contact test) and
positive control (dimeothate) were also included.  Two percent mortality
was observed in the contact toxicity test (4% mortality in the control)
while no mortality was observed in the acute oral toxicity test (2%
mortality in the control).  There were no signs of abnormal behavior in
the surviving honey bees throughout the tests.  The 24 and 48 hour
contact LD50 of Trichoderma asperellum strain ICC 012 was >50 µg
product (2.1 x 105 cfu/bee).  The 24 and 48 hour oral LD50 of
Trichoderma asperellum strain ICC 012 was >111.5 µg product (4.7 x 105
cfu/bee). This study is classified as unacceptable because the study
duration was only 48 hours, which is too short a duration to allow for
detection of pathogenicity to honey bees.  The study is not upgradable
and does not satisfy the data requirement for Tier I testing with honey
bee.

Non-guideline Studies

i) Acute Toxicity (14 Days) of Trichoderma asperellum to the Earthworm
Eisenia fetida in Artificial Soil   (MRID No.: 47345910)

l) corresponding to the highest concentration tested.  The NOEC was
≥1000 mg/kg soil (4.2 x 109 cfu/kg soil).  This non-guideline study is
classified as “ACCEPTABLE.”

ii)  Effects of Trichoderma asperellum to the Activity of Soil
Microflora in the Laboratory (MRID No.: 47345909)

The effects of Trichoderma asperellum on the activity of soil microflora
as determined by carbon mineralization and soil nitrogen transformation
was assessed in a 28-day laboratory study.  Test concentrations of
Trichoderma asperellum strain ICC 012 were 0.07 and 0.67 mg/kg soil dry
weight were applied to loamy, sandy soils collected from a fallow field.
 On Day 28 after application, the respiration rates of Trichoderma
asperellum treated soils were 1.53% and 1.64% lower than in the
untreated control for the test concentrations of 0.07 mg and 0.67 mg
Trichoderma asperellum/kg soil dry weight, respectively, which were not
statistically different from the control, and were below the 25% trigger
values set by the OECD guideline, indicating no long-term effects of the
test substance on soil respiration.  While some significant differences
were observed in soil nitrate content, nitrate formation rate, and
mineral nitrogen soil content, the differences were minor and did not
exceed the 25% trigger values on day 28 according to the OECD guideline
indicating no long-term effects to soil microflora.  This non-guideline
study is scientifically sound and classified as acceptable for assessing
risk of the effects of Trichoderma asperellum strain ICC 012 on soil
microflora.

2.  Environmental Effects Conclusions

a. Terrestrial Animals and Plants

The proposed greenhouse applications for the EP containing Trichoderma
asperellum strain ICC 012 will limit environmental release of the active
ingredient, and exposure to non-target organisms is not expected
following these uses.  Exposure to terrestrial non-target species is
anticipated as a result of some of the proposed use patterns.
Applications made to cuttings and bare roots, in substrate mixes, and
through soil drenches would limit the presence of the active ingredient
to the soil immediately around the treated plants, which will
significantly limit non-target exposure to only those organisms that
inhabit those areas.  The exposure will be limited even further when the
treated plants are in containers (e.g., in pots, flats, etc.).  The
potential for terrestrial non-target exposure would increase with
broadcast applications and chemigation.

The supporting rationale to waive the non-target data requirement for
avian wildlife study was sufficient to conclude that adverse effects are
not expected in avian wildlife as a result of exposure to Trichoderma
gamsii strain ICC 080, due to the growth temperatures required for the
microbial active ingredient compared with the higher body temperature of
birds.

Some strains of Trichoderma harzianum have shown adverse effects on
insects (e.g., Glinski and Buczek 2003, Santamarina et al. 2002).  BPPD
has previously recognized the potential for Trichoderma harzianum T-39
to affect honey bees and non-target insects, particularly coleopterans
(beetles), and required honey bee and ladybird beetle studies for the
registration of T. harzianum T-39 (USEPA 1998, USEPA 2000).
Reclassification of T. harzianum to T. asperellum occurred after some of
these reports were published, and the relatedness of T. asperellum to
these T. harzianum strains is unknown.

The data for toxicity/pathogenicity to honey bees (Apis mellifera) were
unacceptable due to the short duration (48 hours) of the oral and
contact tests that were performed in the study that was submitted.
Honeybee studies should be long enough (e.g., 30 days) to allow for the
observation of latent pathogenic effects.  Because of the
reclassification of Trichoderma harzianum to Trichoderma asperellum, and
the potential effects to honey bees, the product must not be used when
bees are within the area to be treated.  The precautionary label
statement: “Do not apply this product while bees are actively visiting
the treatment area” is included on the proposed EP label and must be
included on any future products containing Trichoderma asperellum as an
active ingredient.  Data or other information would be needed in order
for the Agency to determine that Trichoderma asperellum strain ICC 012
does not pose a hazard to honey bees.

Although not a data requirement for microbial fungicides, the applicant
submitted one insect toxicity/pathogenicity study with the predatory
mite, Typhlodromus pyri.  The study results indicated that adverse
effects on these and related insects are not expected at field
application rates.  However, since Trichoderma harzianum has been shown
to cause effects in a coleopteran insect (Glinski and Buczek 2003,
Santamarina et al. 2002), the following precautionary labeling statement
must be included the proposed EP and future product containing
Trichoderma asperellum strain ICC 012 as an active ingredient:  “This
product may pose a hazard to beneficial coleopteran species. Do not
apply this product within ¼ mile of endangered beetle habitat."  In
order for this label statement to be removed, acceptable data would be
needed to demonstrate that Trichoderma asperellum poses no harm to
coleopteran species.

Non-target plant testing is not required because Trichoderma asperellum
strain ICC 012 is not related to any known plant pathogen.  Adverse
effects on plants are not expected to result from proposed uses of
Trichoderma asperellum strain ICC 012.

Two non-guideline studies on earthworm toxicity/pathogenicity and
effects on soil microflora activity were also submitted.  The 14-day
earthworm (Eisenia fetida) contact LD50 was determined to be >4.2 x 109
cfu/kg soil which is higher than the expected concentration within soil
of 1-cm depth at the highest broadcast application rate on the proposed
label.  No abnormal behavior or effect on body weight was observed.  In
the soil microflora study, soil respiration, nitrate content, nitrogen
formation rate, and mineral nitrogen content were not significantly
perturbed, since all were determined to be below the 25% threshold value
for determining effects.  While these studies are not required for U.S.
registrations of microbial pesticides, they do provide useful
information to additionally conclude that proposed applications of
Trichoderma asperellum strain ICC 012 are not expected to have adverse
effects in soil.

Based on the above data and rationale, adverse effects are not expected
to occur to terrestrial animals or as a result of proposed applications
of Trichoderma asperellum strain ICC 012.

b. Aquatic Animals and Plants

Exposure to Trichoderma asperellum strain ICC 012 in the aquatic
environment is not expected to be significant.  For example, an
application at the highest broadcast application rate (5 lbs/acre) made
directly to a pond that is 6-inches deep (a standard size used by OPP to
estimate aquatic exposure) would yield a concentration of 184 cfu/mL, so
the amount reaching nearby aquatic environments due to runoff or drift
would be expected to be less.  These amounts are far below the maximum
hazard concentrations at which aquatic testing was performed for the
TGAI.  Futhermore, Trichoderma asperellum strain ICC 012 grows in soil,
and if it enters the aquatic environment it is not expected to
proliferate.

Studies with the TGAI were submitted for the freshwater fish and
freshwater invertebrate toxicity/pathogenicity testing data
requirements.  The 30-day LC50 for rainbow trout (Oncorhynchus mykiss)
is >2.4 x 1010 cfu/L.  The 21-day EC50 for Daphnia based on
mortality/immobility was >1.7 x 1010 cfu/L and the NOEC based on
reproduction was 7.6 x 109 cfu/L.  No significant adverse effects were
observed in either study.  All of these endpoint values are above the
maximum hazard dose for aquatic testing with microbial pesticides,
indicating a lack of effects above environmental concentrations that may
occur in aquatic environments as a result of the proposed applications
of Trichoderma asperellum strain ICC 012.  A 48-hour Daphnia test with
the EP was submitted but was determined to be unacceptable due to the
inadequate study duration; however, the available data on the TGAI are
adequate to assess the potential risk of Trichoderma asperellum strain
ICC 012.  Trichoderma species are ubiquitous in the environment, and do
not proliferate in aquatic ecosystems, so significant exposure in
freshwater environments is not expected from the proposed applications
of Trichoderma asperellum strain ICC 012.  Therefore, adverse effects to
freshwater aquatic animals are not anticipated.

Trichoderma species are not related to any known plant pathogens, and as
determined above, Trichoderma asperellum strain ICC 012 is not expected
to occur in freshwater environments in significant amounts.  Therefore,
data from aquatic plant testing are not required, and no effects are
anticipated to this taxon as a result of the proposed applications.  A
study with the aquatic vascular plant, Lemna gibba, was submitted and
reviewed, and results show that the 7-day growth inhibition NOEC for the
aquatic vascular plant Lemna gibba is 1.7x1011 cfu/L.  These results
confirm the conclusion of no anticipated adverse effects to aquatic
plants.

As with freshwater environments, significant amounts of T. asperellum
strain ICC 012 are not expected to reach marine/estuarine environments.
Therefore data are not required for marine/estuarine non-target species,
and adverse effects in these environments are not anticipated.

Based on the data submitted and what is known about the nature of
Trichoderma species in aquatic environments, adverse effects to
freshwater and marine/estuarine fish, invertebrates, and plants are not
expected as a result of exposure following proposed labeled applications
of T. asperellum strain ICC 012.

3. Threatened and Endangered Species Assessment

Direct Effects

BPPD has made a “No Effect” determination for direct effects to
listed species.

There are no listed endangered or threatened species related to the
target fungal pests.  Since it is concluded that effects are not
anticipated for avian wildlife, wild mammals, terrestrial or aquatic
plants, or freshwater or marine/estuarine aquatic animals, the proposed
applications of Trichoderma asperellum strain ICC 012 are not expected
to result in direct effects to listed species in these taxa.

However, the U.S. Fish and Wildlife Service lists sixteen endangered
coleopteran beetle species.  Fifteen of the endangered and threatened
beetles only occur in caves or aquatic habitats.  None of these
endangered beetles inhabit or are expected to occur in or near
agricultural fields when Trichoderma asperellum strain ICC 012 is used
according to label directions.  One endangered coleopteran insect, the
American burying beetle, may occur in old fields or cropland hedge rows.
 However, based upon the feeding habits of the American burying beetle,
its larvae are not going to be exposed to Trichoderma asperellum strain
ICC 012 from its use as a microbial fungicide.  Adult American burying
beetles are opportunistic scavengers that feed on dead animal tissues.
They bury vertebrate carcasses in soil and lay their eggs in the
carcass.  Carrion regurgitated by adults is fed to the larvae until they
are able to feed directly on the carcass. (  HYPERLINK
"http://www.fws.gov/endangered/wildlife.html"
http://www.fws.gov/endangered/wildlife.html )  Therefore no exposure of
this beetle to Trichoderma asperellum strain ICC 012 used as a microbial
fungicide would occur

Even though overlap of endangered coleopteran insect habitats with the
proposed use sites is not expected, as an added precaution the following
statement must be included on labels of the proposed EP and future
products containing Trichoderma asperellum strain ICC 012 as an active
ingredient: “Do not apply this product within ¼ mile of endangered
beetle habitat.”  With the requisite precautionary statements
described previous for honey bees, and with consideration of the lack of
effects to the other taxa listed above, BPPD does not anticipate direct
effects to listed species from the proposed uses of Trichoderma
asperellum strain ICC 012 as a microbial fungicide.

Indirect Effects and Critical Habitat

The proposed uses of Trichoderma asperellum strain ICC 012 are not
expected to result in indirect effects to listed species or effects on
their designated critical habitats as a result of losses of terrestrial
or aquatic animals or plants.  Trichoderma harzianum has not shown
effects to a broad range of insect species, anddata reviewed by BPPD
demonstrated that Trichoderma asperellum strain ICC 012 is not toxic or
pathogenic to some insects.  There are no listed species known to be
exclusively dependent on coleopteran beetles, and the Agency does not
expect a significant reduction in insect populations in general to occur
in areas that Trichoderma asperellum strain ICC 012 is used as a
microbial fungicide.  The precautionary label statements for bees
described above (Section IV(C)(2)(a)) are expected to adequately protect
bees where Trichoderma asperellum strain ICC 012 is applied, and
indirect effects to limited species’ critical habitats due to loss or
reduction of bee populations are not anticipated.  Therefore, BPPD makes
a “No Effect” determination for indirect effects to listed species.

For a comprehensive guideline-by-guideline summary of the non-target
toxicity data requirements, refer to   HYPERLINK  \l "Table4"  Table 3
in Appendix A

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 Trichoderma asperellum strain ICC
012, compared to the general population.  Please comment if you are
aware of any sub-populations that may have atypical, unusually high
exposure compared to the general population.

VI.   RISK MANAGEMENT AND REGISTRATION DECISIONS tc "V.   RISK
MANAGEMENT AND REGISTRATION DECISION"

	A.  Determination of Eligibility

 tc "A.  Determination of Eligibility " \l 2

Section 3(c)(5) of FIFRA provides for the registration of new active
ingredients if it is determined that (A) its composition is such as to
warrant the proposed claims for it; (B) its labeling and other materials
required to be submitted comply with the requirements of FIFRA; (C) it
will perform its intended function without unreasonable adverse effects
on the environment; and (D) when used in accordance with widespread and
commonly recognized practice it will not generally cause unreasonable
adverse effects on the environment.

The four criteria of the Eligibility Determination for Pesticidal Active
Ingredients are satisfied by the science assessments supporting products
containing Trichoderma asperellum strain ICC 012.  Such products are not
expected to cause unreasonable adverse effects, and are likely to
provide protection as claimed when used according to label instructions.
Therefore, Trichoderma asperellum strain ICC 012 is eligible for
registration for the labeled uses.

B.  Regulatory Decision

On October 1, 2009, EPA announced a new policy to provide a more
meaningful opportunity for the public to participate on major
registration decisions before they occur.  According to this new policy,
EPA intends to provide a public comment period prior to making a
registration decision for, at minimum, the following types of
applications:  new active ingredients; first food use; first outdoor
use; and first residential use.  Accordingly, this pesticide is  subject
to a 30-day comment period as a new active ingredient with both outdoor
uses and residential uses.  Any regulatory decision will be informed by
the comments EPA receives.

At this time, EPA believes, the data submitted fulfill the requirements
of registration for products contatining Trichoderma asperellum strain
ICC 012 as a microbial fungicide.  Acute toxicity data for Trichoderma
asperellum strain ICC 012 demonstrate that it has low acute toxicity
(category III or IV).  EPA has no concerns for any non-target organisms,
including threatened or endangered species, exposed to Trichoderma
asperellum strain ICC 012 when it is used in accordance with approved
label directions.  EPA has not identified any toxic endpoints for
non-target mammals, birds, honey bees, non-target insects, plants,
aquatic, or soil organisms.  EPA supports its registration under Section
3(c) (5) of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA).  Refer to Appendix B for product-specific information.

		1.  Conditional/Unconditional Registration

The Environmental Protection Agency (EPA) considered information
submitted for granting registration under Section 3(c)(5) of the Federal
Insecticide, Fungicide and Rodenticide Act (FIFRA) and determined that
the data and information submitted in support of Trichoderma asperellumi
strain ICC 012 adequately satisfy current data requirements (refer to 40
CFR Subpart U § 158.2000).  If the Agency receives comments during the
30 day public comment period that inform EPA’s initial decision, EPA
will address such new information and take appropriate action.

		C.  Labeling

Before releasing pesticide products containing Trichoderma asperellum
strain ICC 012 for shipment, the applicant is required to provide the
Agency with appropriate labels.

VII.   ACTIONS REQUIRED BY THE APPLICANT

The Agency evaluated the data submitted in connection with the initial
registration of  Trichoderma asperellum strain ICC 012 and determined
that these data fulfill current registration guideline requirements.  No
additional data are required to be submitted to the Agency at this time.
 Additional data may be required for new uses and/or changes to existing
uses.

Not withstanding the information stated in the previous paragraph, it
should be clearly understood that certain, specific, data are required
to be reported to the Agency as a requirement for maintaining the
Federal registration for a pesticide product. A brief summary of these
types of data are listed below.

A.  Reporting of Adverse Effects and Hypersensitivity Incidents

Reports of all incidents of adverse effects to the environment must be
submitted to the Agency under the provisions stated in FIFRA, Section
6(a)(2).

Additionally, all incidents of hypersensitivity (including both
suspected and confirmed incidents) must be reported to the Agency under
the provisions of 40 CFR Part 158.2140 (Guideline reference number OPPTS
885.3400).

VIII.  GLOSSARY OF ACRONYMS AND ABBREVIATIONS

BCCM	Belgian Coordinated Collections of Microorganisms

BPPD	Biopesticides and Pollution Prevention Division

BRAD	Biopesticides Registration Action Document

CFR	Code of Federal Regulations

cfu 	colony-forming unit

cm2 	square centimeter

°C 	degrees Celsius

EDSP	Endocrine Disruptor Screening Program

EDSTAC	Endocrine Disruptor Screening and Testing Advisory Committee

EP	end-use product

EPA	Environmental Protection Agency (the “Agency”)

FFDCA	Federal Food, Drug, and Cosmetic Act

FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act

FQPA 	Food Quality Protection Act

FR	Federal Register

g 	gram

kg	kilogram

L 	Liter

LC50	median lethal concentration. A statistically derived concentration
of a

	substance that can be expected to cause death in 50% of test animals.
It is

	usually expressed as the weight of substance per weight or volume of
water,

愉物牯映敥攨朮Ⱞ洠⽧ⱌ洠⽧杫牯瀠浰⸩䰍䍌ठ
楬業整楬扡汩瑩⁹潣灭湡൹佌䍅氉睯獥⁴扯敳癲扡敬
攠晦捥⁴潣据湥牴瑡潩൮前䑉丠⹯䴉獡整⁲敒潣摲䤠敤
瑮晩捩瑡潩畎扭牥

μm 	micrometer

mg	milligram

mL	milliliter

mm	millimeter

MUCL 	Mycothèque de l'Université Catholique de Louvain

NIOSH 	National Institute for Occupational Safety and Health

NOEC	no observable effect concentration

OPP	Office of Pesticide Programs

OPPTS 	Office of Prevention, Pesticides, and Toxic Substances

PII 	Primary Irritation Index

P.O. Box	Post Office Box

PP 	Pesticide Petition

PPE 	personal protective equipment

TGAI 	technical grade of the active ingredient

IX.  BIBLIOGRAPHY

A. Studies Submitted in Support of Trichoderma asperellum strain ICC 012

MRID	Citation Reference

47345900	Isagro USA, Inc. (2008) Submission of Product Chemistry, Fate
and Toxicity Data in Support of the Application for Registration of
Trichoderma asperellum ICC 012 Technical. Transmittal of 13 Studies.

47345901	Leuschner, P. (2004) Acute Toxicity Study of Trichoderma
harzianum Strain ICC 012 by Oral Administration to Rats. Project Number:
17792/04. Unpublished study prepared by Laboratory of Pharmacology &
Toxicology. 16 p.

47345902	Leuschner, P. (2004) Acute Toxicity Study of Trichoderma
harzianum Strain ICC 012 by Intraperitoneal Injection to Rats. Project
Number: 17794/04. Unpublished study prepared by Laboratory of
Pharmacology & Toxicology . 27 p.

47345903	Leuschner, P. (2004) Acute Pulmonary Toxicity / Pathogenicity
Study of Trichoderma harzianum ICC 012 by Intratracheal Administration
to Rats. Project Number: 17793/04. Unpublished study prepared by
Laboratory of Pharmacology & Toxicology. 45 p.

47345904	Dengler, D. (2004) Analysis of the Occurrence of Test Substance
Trichoderma harzianum Strain ICC 012 in Animal Tissue. Project Number:
20041113/01/AMAT. Unpublished study prepared by GAB Biotechnologie Gmbh.
23 p.

47345905	Pawlowski, S. (2004) Toxicity of Trichoderma harzianum Strain
ICC 012 to Rainbow Trout (Oncorhynchus mykiss) in a Prolonged Toxicity
Test. Project Number: 17103231. Unpublished study prepared by Institut
fuer Biologische Analytik und Consulting IBACON. 64 p.

47345906	Pawlowski, S. (2004) Influence of Trichoderma harzianum Strain
ICC 012 to Daphnia magna in a Reproduction Test. Project Number:
17102221. Unpublished study prepared by Institut fuer Biologische
Analytik und Consulting IBACON. 64 p.

47345907	Schmitzer, S. (2004) Effects of Trichoderma harzianum (Acute
Contact and Oral) on Honey Bees (Apis mellifera L.) in the Laboratory.
Project Number: 17105035. Unpublished study prepared by Institut fuer
Biologische Analytik und Consulting Ibacon. 27 p.

47345908	Spatz, B. (2004) Toxicity of Trichoderma harzianum Strain ICC
012 to the Aquatic Plant Lemna gibba in a Growth Inhibition Test.
Project Number: 17104240. Unpublished study prepared by Institut fuer
Biologische Analytik und Consulting IBACON. 69 p.

47345909	Reis, K. (2004) Effects of Trichoderma harzianum on the
Activity of the Soil Microflora in the Laboratory. Project Number:
17109080. Unpublished study prepared by Institut fuer Biologische
Analytik und Consulting IBACON. 48 p.

47345910	Luhrs, U. (2004) Acute Toxicity (14 Days) of Trichoderma
harzianum to the Earthworm Eisenia fetida in Artificial Soil. Project
Number: 17108021. Unpublished study prepared by Institut fuer
Biologische Analytik und Consulting IBACON. 25 p.

47345911	Winkler, V. (2007) Trichoderma asperellum Strain ICC 012
(Formerly Trichoderma harzianum): Group A: Product Analysis. Project
Number: 110307. Unpublished study prepared by ISAGRO S.p.A. 56 p.

47345912	Winkler, V. (2007) Trichoderma asperellum Strain ICC 012
(formally Trichoderma harzianum): Group B Physical and Chemical
Properties (Self Certification). Project Number: 112707. Unpublished
study prepared by Chemservice S.r.l. 7 p.

47345913	Garofani, S. (2007) Trichoderma harzianum: Complete Analysis of
Five Batch Samples. Project Number: CH/315/2007. Unpublished study
prepared by Chemservice S.r.l. 28 p.

47346300	Isagro S.P.A. (2008) Submission of Product Chemistry and
Toxicity Data in Support of the Application for Registration of Bioten
WP (Remedier WP). Transmittal of 10 Studies.

47346301	Leuschner, P. (2004) Acute Toxicity Study of Remedier WP
(Trichoderma harzianum and Trichoderma viride Wettable Powder) by Oral
Administration to Rats. Project Number: 17795/04. Unpublished study
prepared by Laboratory of Pharmacology & Toxicology . 30 p.

47346302	Leuschner, P. (2004) Acute Toxicity Study of Remedier WP
(Trichoderma harzianum and Trichoderma viride Wettable Powder) in Rats
by Dermal Administration. Project Number: 17796/04, 140185. Unpublished
study prepared by Laboratory of Pharmacology & Toxicology . 33 p.

47346303	Leuschner, P. (2004) Acute Inhalation Toxicity Study of
Remedier WP (Trichoderma harzianum and Trichoderma viride Wettable
Powder) by Oral in Rats. Project Number: 17797/04, 140185. Unpublished
study prepared by Laboratory of Pharmacology & Toxicology . 35 p.

47346304	Leuschner, P. (2004) Acute Eye Irritation Study of Remedier WP
(Trichoderma harzianum and Trichoderma viride Wettable Powder) by
Instillation into the Conjunctival Sac of Rabbits. Project Number:
17799/04, 140185. Unpublished study prepared by Laboratory of
Pharmacology & Toxicology. 27 p.

47346305	Leuschner, P. (2004) Acute Skin Irritation Test (Patch Test) of
Remedier WP (Trichoderma harzianum and Trichoderma viride Wettable
Powder) in Rabbits. Project Number: 17798/04, 140185. Unpublished study
prepared by Laboratory of Pharmacology & Toxicology . 27 p.

47346306	Leuschner, P. (2004) Examination of Remedier WP (Trichoderma
harzianum and Trichoderma viride Wettable Powder) in a Skin
Sensitisation Test in Guinea Pigs According to Magnusson and Kligman
(Maximisation Test). Project Number: 17800/04, 140185. Unpublished study
prepared by Laboratory of Pharmacology & Toxicology . 40 p.

47346307	Pawlowski, S. (2004) Acute Toxicity of Remedier WP to Daphnia
magna in a 48-hour Immobilization Test. Project Number: 19561220.
Unpublished study prepared by Institut fuer Biologische Analytik und
Consulting IBACON. 46 p.

47346308	Rosenkranz, B. (2004) Effects of Remedier WP on the Predatory
Mite Typhlodromus pyri in the Laboratory - Dose Response Test. Project
Number: 19562063. Unpublished study prepared by Institut fuer
Biologische Analytik und Consulting IBACON. 40 p.

47346309	Winkler, V. (2007) Bioten WP: Group A: Product Analysis.
Project Number: 111907. Unpublished study prepared by Isagro S.P.A. 50
p.

47346310	Winkler, V. (2007) Bioten WP: Group B: Physical and Chemical
Properties (Self Certification). Project Number: 112607. Unpublished
study prepared by Chemservice S.P.A. 7 p.

47460900	Isagro USA, Inc. (2008) Submission of Product Chemistry Data in
Support of the Applications for Registration of Trichoderma gamsii ICC
080 Technical, Trichoderma asperellum ICC 012 Technical, and Bioten WP.
Transmittal of 1 Study.

47460901	Winkler, V (2008) Response to EPA April 24, 2008 Letter
"Deficiencies Noted During Preliminary Screen of Submissions for
Trichoderma gamsii ICC 080 Technical: Trichoderma asperellum ICC 012
Technical: and Bioten WP" . Project Number: 062308. Unpublished study
prepared by Chemservice S.P.A., DSMZ-Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, and Isagro S.p.A. Unita prodotti
Biologic. 88 p.

47643900	Isagro S.P.A. (2009) Submission of Product Chemistry Data in
Support of the Application for Registrations of Bioten WP, Trichoderma
Gamsii ICC 080 Technical and Trichoderma asperellum ICC 012 Technical.
Transmittal of 1 Study.

47643901	Lodini, S. (2008) Mesophilic Contaminants in Active Ingredients
Trichoderma gamsii Strain ICC 080 and Trichoderma asperellum ICC 012:
Final Report. Project Number: REM/80122. Unpublished study prepared by
Isagro S.P.A. 30 p.

47802700	ISAGRO S.P.A. (2009) Submission of Product Chemistry Data in
Support of the Applications for Registration of Trichoderma gamsii ICC
080 Technical, Trichoderma asperellum ICC 012 Technical, and Tenet WP.
Transmittal of 1 Study.

47802701	Graben, M. (2009) Response to EPA on Phone Conference of June
4, 2009 and April 2, 2009 Letter: Deficiencies Noted During Review of
Submissions for Trichoderma gamsii ICC 080 Technical: Trichoderma
asperellum ICC 012 Technical: and Bioten WP (EPA File Symbols: 80289-RN:
and 8028-O [PRIA Category: B590]. Including Two Updated End Use Labels
and Refined CSF. Project Number: 061209, 901, 800/02REV. Unpublished
study prepared by ISAGRO S.P.A. 88 p.

B.  Environmental Protection Agency Risk Assessment Memorandums

U.S. EPA.  1998.  Non-target organism data requirements for Makhtashim
Chemical Works Ltd.’s Trichodex containing the microbial fungicide
Trichoderma harzianum T-39.  Memorandum from M. Mendelsohn to S. Bacchus
and P. Hutton.

U.S. EPA. 2000.  Trichoderma harzianum Rifai strain T-39 (119200)
technical document.  URL:   HYPERLINK
"http://epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/tech_119200
.htm"
http://epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/tech_119200.
htm .

U.S. EPA 2008.  Trichoderma species final Reregistration Review
decision.  Case 6050.  Docket No. EPA-HQ-OPP-2006-0245.

C. Other References Cited

Glinski, Z. and K. Buczek.  2003.  Response of the Apoidea to fungal
infections.  Apiacta 38:183-189.

Gouli, S., V. Gouli, M. Skinner, B. Parker, J. Marcelino, and M.
Shternshis.  2008.  Mortality of Western flower thrips (Frankliniella
occidentalis) under influence of single and mixed fungal inoculations.
Journal of Agricultural Technology 4(2):37-47.

Howell, C. R.  2003.  Mechanisms employed by Trichoderma species in the
biological control of plant diseases: the history and evolution of
current concepts.  Plant Disease 87(1):4-10.

Prinzinger, R., A. Pressmar, and E. Schleucher.  1991.  Body temperature
in birds.  Comparative Biochemistry and Physiology 99A(4):499-506.

Santaramarina, M. P., J. Rosello, R. Llacer, and V. Sanchis.  2002.
Antagonistic activity of Penicillium oxalicum Corrie and Thom,
Penicillum decumbens Thom, and Trichoderma harzianum Rifai isolates
against fungi, bacteria and insects in vitro.  Revista Iberoamericana de
Micologia 19:99-103.

Jijakli MH, Lepoivre P, Tossut P, Thornard P. 1993. Biological control
of Botrytis

		cinerea and Penicillium sp. on post-harvest apples by two antagonistic
yeasts.

		Med. Fac. Landbouww. Univ. Gent 53(3b):1349–1358.

  HYPERLINK "http://www.fws.gov/endangered/wildlife.html"
http://www.fws.gov/endangered/wildlife.html   U.S. Fish and Wildlife
Service    Endangered Species Program Website

Yemma JJ, Berk MP. 1994. Chemical and physiological effects of Candida
albicans

		toxin on tissues. Cytobios 77(310):147–158.

APPENDIX A – MICROBIAL PESTICIDE DATA REQUIREMENTS

TABLE 1. Product Analysis Data Requirements for the Technical Grade of
the Active Ingredient (TGAI),

                  Trichoderma  asperellum strain ICC 012, and Its
Associated End-Use Product (EP), Tenet WP (40 CFR § 158.2120)

Data Requirement

 (OPPTS Guideline)	Results	MRID Number

	TGAI	EP

	Product Chemistry and Composition

Product Identity

 (885.1100)	The strain ICC 012, originally characterized as Trichoderma
harzianum, has been recharacterized as Trichoderma asperellum and also
has been characterized by molecular biology techniques.  Submitted data
satisfy the requirements

Classification: Acceptable 	Submitted data satisfy the requirements of
Product Identity for the EP.

  Classification: Acceptable	47345911

47346309

47460901

47643901

Manufacturing Process (885.1200)	Submitted data satisfy the requirements
of manufacturing process for both the TGAI and EP.

Classification: Acceptable	47345911

47346309

47460901

Deposition of a Sample in a Nationally Recognized Culture Collection

(Not applicable)	Trichoderma asperellum strain ICC 012 has been
deposited in the CABI Bioscience International Mycological
Institute-Egham, UK with the Accession Number IMI CC No. 392716.

	Discussion of Formation of Unintentional Ingredients (885.1300)
Submitted data satisfy the requirements of discussion of formation of
unintentional ingredients for both the TGAI and EP.

Classification: Acceptable	47345911

47346309

47460901

47802701

Analysis and Certified Limits

Analysis of Samples (885.1400)	Submitted data satisfy the requirements
of analysis of samples for both the TGAI and EP.

Classification: Acceptable	47345911

47345913

47346309

47460901

    47802701

Certification of Limits (885.1500)	The certified limits for the

active ingredient were within the

specified range.

Classification: Acceptable 	 The certified limits for the

 active ingredients exceed the

 OPPTS Guideline 830.1750

 specified ranges, but an

 acceptable explanation was

 provided.

 Classification: Acceptable	47345911

47346309

47460901

47802701

Physical and Chemical Characteristics

Color

(830.6302)	Gray green 	Not applicable	47345912

47346310

Physical State

(830.6303)	Solid powder	Not applicable	47345912

47346310

Odor

(830.6304)	Slight odor	Not applicable	47345912

47346310

Stability to Normal and Elevated Temperatures, Metals, and Metal Ions

(830.6313)	Not required for MP

	Not applicable	47345912

47346310

Storage Stability

(830.6317)	Stable for 6 months at 20ºC, not stable at 54ºC	Active
ingredient stable for 15 months at 20-25ºC

	47345912

47346310

Miscibility

(830.6319)	Product is not a suspension.	Product is not a suspension.

	47345912

Corrosion Characteristics

(830.6320)	Product is not corrosive.	Product is not corrosive.	47345912

pH

(830.7000)	 6.21 (1% aqueous solution)	Not applicable	47345912

.

Viscosity

(830.7100)	Product is not a liquid. 	Product is not a liquid. 	47345912

47346310

Density/Relative Density/Bulk Density

(830.7300)	0.195 g/mL.	Not applicable	47345912

47346310

TABLE 2. Toxicology Data Requirements for the Technical Grade of the
Active Ingredient (TGAI), Trichoderma asperellum strain ICC 012, and Its
Associated End-Use Product (EP), Bioten WP(40 CFR § 158.2140)

Data Requirement

(OPPTS Guideline)	Results	MRID Number

	TGAI	EP

	Tier I

Acute Oral Toxicity/Pathogenicity

(885.3050)	Trichoderma asperellum  strain ICC 012 (4.2 x 109 CFU/g in
0.9% NaCl solution) was not toxic in rats at a dose of  2000 mg/kg bw

This study was rated SUPPLEMENTAL but was upgraded to “ACCEPTABLE”
by clearance performed in the pulmonary toxicity/pathogenicity study.

Classification: Acceptable	Not applicable

47345901

Acute Pulmonary Toxicity/Pathogenicity

(885.3150)	Not toxic, infective, and/or pathogenic to rats by pulmonary
dose of

1 x 107 CFU/animal.

Classification: Acceptable	Not applicable	47345903 47345904

Acute Intraperitoneal Injection Toxicity/Pathogenicity

(885.3200)	Trichoderma asperellum strain ICC 012 (4.2 x 109 CFU/g) in
0.9% NaCl solution was not toxic to rats injected at a dose of  1 x 108
CFU/g in a limit test.  This study was rated SUPPLEMENTAL but was
upgraded to “ACCEPTABLE” by clearance performed in the pulmonary
toxicity/pathogenicity study.

Classification: Acceptable	Not applicable	47345902

Hypersensitivity Incidents

(885.3400)	Any hypersensitivity incidents must be reported per OPPTS
Guideline 885.3400.

	Cell Culture

(885.3500)	Not required because Trichoderma asperellum  strain ICC 012
is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)).	Not
applicable	Not applicable

Acute Oral Toxicity

(870.1100)

	Not applicable	Bioten WP/ Remedier WP (Trichoderma asperellum  7.8 x
107 CFU/g; Trichoderma gamsii 4.2 x 107 CFU/g) is not toxic 5.3.9) in
the rat.

Oral LD50  was

> 2000 mg/kg bw

Limit test; no mortality occurred during the study

Classification: Acceptable

TOXICITY CATEGORY III 	47346301

Acute Dermal Toxicity

(870.1200)	Not applicable	When dosed with Bioten WP/ Remedier WP in 0.9%
NaCl at a dose of 2000 mg/kg bw is of MODERATE Toxicity.  There were no
treatment related clinical signs, necropsy findings or changes in body
weight.

Classification: Acceptable

TOXICITY CATEGORY III 	43746302

Acute Inhalation Toxicity

(870.1300)	Not applicable	 Bioten WP/ Remedier WP Inhalation LC50were as
follows: Males > 5.20 mg/L,

Females > 5.20 mg/ L, Combined > 5.20 mg/L.

Bioten WP/ Remedier WP is of LOW Toxicity, based on lack of mortality in
male and female rats. There were no treatment related clinical signs,
necropsy findings or changes in body weight.

Classification: Acceptable

TOXICITY CATEGORY IV 	47346303

Acute Eye Irritation

(870.2400)	Not applicable	Bioten WP was not an eye irritant.

Classification: Acceptable

TOXICITY CATEGORY IV 	47346304

Primary Dermal Irritation

(870.2500)	Not applicable	When dosed with 500 mg of Bioten WP/ Remedier
WP (Trichoderma conidia (1.2 x 108 CFU/g); Trichoderma asperellum strain
ICC 012 (7.8 x 107 CFU/g); Trichoderma gamsii strain ICC 080 (4.2 x 107
CFU/g)), the product is slightly irritating.

Classification: Acceptable

TOXICITY CATEGORY IV	47346305

Skin Sensitization

(870.2600)	Not applicable	This study is not required

Bioten WP was not a dermal sensitizer.

Classification: Acceptable	47346306

Tiers II and III

Not required for Trichoderma asperellum strain ICC 012  based on the
lack of acute toxicity/pathogenicity in the Tier I studies.

TABLE 3.   Summary of data submitted to comply with non-target organism
data requirements published in 40 CFR § 158.2150 for support of the
registration of Trichoderma asperellum strain ICC 012

Data Requirement	OPPTS Guideline No.	Results Summary and Classification
MRID No.

Avian oral toxicity	885.4050	Data waiver rationale provides sufficient
information to determine that toxicity/pathogenicity to avian wildlife
is not expected.

Classification:  Acceptable	Contained in tolerance petition

Avian inhalation toxicity/pathogenicity	885.4100	Data are not required.
Nature of microbial pesticide does not indicate potential pathogenicity
to birds or relatedness to any known bird pathogens.	N/A

Wild mammal toxicity/pathogenicity	885.4150	Tests required by 40 CFR §
158.2140 are adequate and appropriate for assessment of hazards to wild
mammals.  Testing indicates no adverse effects to laboratory rats at 4.3
x 108 spores/mL (2000 mg TGAI/kg bw) when dosed orally.  Testing with
the EP indicates no adverse effects to laboratory rats dosed orally at
2.3 x 107 spores/mL (2000 mg EP/kg bw).

Classification:  Acceptable for wild mammal risk assessment	47345901

47346301

Freshwater fish toxicity/pathogenicity	885.4200	The 30-day LC50 for
rainbow trout (Oncorhynchus mykiss) is >2.4 x 1010 cfu/L.

Classification:  Acceptable 	47345905

Freshwater invertebrate toxicity/pathogenicity	885.4240	The 21-day EC50
for Daphnia based on mortality/immobility was >1.7 x 1010 cfu/L and the
NOEC based on reproduction was 7.6 x 109 cfu/L.  A 48-hour Daphnia test
with the EP was submitted but was determined to be unacceptable due to
the inadequate study duration.

Classification:  Acceptable	47345906

47346307

Estuarine/marine fish and invertebrate testing	885.4280	Data are not
required.  T. asperellum strain ICC 012 will not be applied directly to
water and is not expected to enter marine/estuarine environments in
amounts that are significantly higher than naturally-occurring
concentrations.	N/A

Non-target plant testing	885.4300	Data are not required, since T.
asperellum strain ICC 012 is not related to known plant pathogens, and
is not expected to have adverse effects on plants.  Additionally, a
7-day growth inhibition NOEC for the aquatic vascular plant Lemna gibba
was 1.7 x1011 cfu/L.

Classification:  Acceptable	47345908

Non-target insect testing	885.4340	LD50 >5 kg/ha for EP (containing T.
gamsii strain ICC 080 and T. asperellum strain ICC 012) for predatory
mite (Typhlodromus pyri) and no significant effects on reproduction
observed.

Classification:  Acceptable	47346308

Honey bee testing	885.4380	48-hour contact and oral LD50 were >2.1 x 105
cfu/bee and >4.7 x 105 cfu/bee, respectively.  The study was of too
short a duration to determine pathogenicity.

Classification:  Unacceptable 	47345907

Non-guideline testing

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ਁ氃愀϶﹐瑹壙_ࠀ≥4.2 x 109 cfu/kg soil.

Classification: Acceptable 	47345910

Non-guideline testing

Effects on Soil Microflora

Activity

N/A	Soil respiration, nitrate content, nitrogen formation rate, and
mineral nitrogen content were below the 25 % threshold value, indicating
no significant perturbation of the soil microflora community

Classification: Acceptable

47345909

APPENDIX B – Trichoderma asperellum strain ICC 012  Products

EPA Registration Number	Registration Name	Percentage Active Ingredient
Formulation Type	Use Site	Method of Application	Application Rate	Target
Pests

80289-9	Bioten  WP

 (or TenetWP)	2.0%

T. gamsii

2.0%

T asperellum	Wettable Powder	Alfalfa

Berries

Cereal Grains

Citrus:

Clover

Cole Crops

Corn:

Cotton

Cucurbits

Grass, Forage, Fodder, and Hay

Fruiting Vegetables

Ginseng

Herbs (fresh, dried and for oil)

Leafy Vegetables (except Brassica) Legume Vegetables

Olive

Onions

Dry bulb

Pineapple

Peanut

Pomegranate Root , Tuber, and Corm Vegetables Sunflower

Tobacco

Tree Fruits

Tree Nuts

Tropical Fruit

At Greenhouse, nursery and field sites

Vines:

(table grapes, winegrapes, muscadines, hops, kiwi)	In-furrow and banded
application.

Broadcast application

Cutting and bare root application

Drip, drench and chemigation.

.

Substrate mix.	Rates vary by crop and application method	Armillaria sp.,
Fusarium spp., Phytophthora spp., Pythium spp., Rhizoctonia spp.,
Rosellinia sp., Sclerotinia spp. Sclerotium rolfsii, Thielaviopsis
basicola, Verticillium spp

80289-11	Trichoderma asperellum strain  ICC 012 	99.9%

Formulating use only	N/A	N/A

Trichoderma asperellum strain ICC 012

Biopesticides Registration Action Document (BRAD)	December 2009

 PAGE

 PAGE   37