SUBDIVISION M
of the
PESTICIDE TESTING
GUIDELINES
a* ana
•/&M v
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March, 1989
PESTICIDE ASSESSMENT GUIDELINES
Subdivision M
Microbial Pest Control Agents
and
Biochemical Pest Control Agents
Prepared by
Janet Andersen, Ph.D.
Defora F. Edwards, Ph.D.
William J. Hazel, Ph.D.
Morris A. Levin, Ph.D.
Robert W. Pilsucki, Ph.D.
William R. Schneider, Ph.D.
Roy D. Sjoblad, Ph.D.
Subdivision M Revision Coordinator
William R. Schneider, Ph.D.
Ecological Effects and Fate Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
Office of Pesticide and Toxic Substances
Washington, D.C. 20460
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FOREWORD
Subdivision M describes protocols which may be used to perform
testing on biochemical and microbial pest control agents to support their
registration as pesticides under the Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA) . Protocols are provided for deterinining the
fate of these pesticides in the environment and for evaluating their
potential adverse effects on humans and other nontarget organisms. Sub-
division M is a nonregulatory companion to 40 CFR Part 158, Data
for Registration. This revision of Subdivision M includes
changes to the data requirements to be incorporated in the next revision
to 40 CFR Part 158.
ACKNGWTFTX5EMENTS
This revision to Subdivision M was reviewed and edited by Amy S.
Rispin, Frederick S. Betz and Zigfridas Vaituzis of the Emdronmental
Fate and Effects Division and by Patricia Roberts of the EPA Office of
General Counsel. The EPA Office of Research and Development provided
scientific support through the microbial pesticide projects at the Health
Effects Research Laboratory/ RTP, North Carolina, under Clint Kawanishi,
and the Environmental Research Laboratory, Corvallis, Oregon, under Raymond
Seidler.
This printing is Part A, Microbial Pest Control Agents. Part B,
Biochemical Pest Control Agents, will be released separately.
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SUBDIVISION M: GUIDELINES FOR TESTING KECROBIAL AND BIOCHEMICAL PEST
CONTROL AGENTS
Table of Contents
INTRODUCTION Page
I. Contents of Subdivision M 1
II. Scope of Subdivision M 1
III. Background of Subdivision M 3
IV. Relationship to Other Subdivisions of the Guidelines 5
SECTION A; MICROBIAL PEST CONTROL APENTS
150A GENERAL INFORMATION 7
150A-1 Overview 7
150A-2 Definitions 10
150A-3 General Provisions 13
150A-4 Reporting of Data 17
151A PRODUCT ANALYSIS GUIDELINES FOR MICROBIAL PEST
CONTROL AGENTS 22
151A-1 Overview 22
151A-2 Through 151-9 [Reserved] 22
151A-10 Product Identity and Disclosure of Ingredients 22
151A-11 Manufacturing Process 24
151A-12 Discussion of Formation of Unintentional
Ingredients 25
151A-13 Analysis of Samples 26
151A-14 [Reserved] 26
151A-15 Certification of Ingredient Limits 26
151A-16 Physical and Chemical Properties 27
151A-17 Submittal of Samples 27
152A TOXICOLOGY GUIDELINES FOR MICROBIAL PEST CONTROL AGENTS 29
152A-1 Overview 29
152A-2 Through 152A-9 [Reserved] 33
Tier I Testing
152A-10 Acute Oral Toxicity/Pathogenicity Study 33
152A-11 Acute Dermal Toxicity Study 39
152A-12 Acute Pulmonary Toxicity/Pathogenicity Study 44
152A-13 Acute Intravenous Toxicity/Pathogenicity Study 50
152A-14 Primary Eye Irritation/Infection Study 56
152A-15 Hypersensitivity Incidents 62
152A-16 Cell Culture Tests with Viral Pest Control
Agents 63
152A-17 Through 152A-19 [Reserved] 68
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11
Tier II Testing
152A-20 Acute Toxicity Study 69
152A-21 Subchronic Toxicity/Pathogenicity Studies 70
Tier III
152A-30 Reproductive and Fertility Effects 75
152A-31 Qncogenicity Study 79
152A-32 Immunodeficiency Studies 79
152A-33 Primate Infectivity/Pathogenicity Study 80
153A RESIDUE ANALYSIS GUIDELINES FOR MICROBIAL PEST CONTROL
AGENTS 81
153A-1 Overview 81
153A-2 [Reserved] 82
153A-3 General Residue Data Requirements 83
153A-4 Chemical Identity 83
153A-5 Directions for Use 84
153A-6 Nature of the Residue in Plants 84
153A-7 Nature of the Residue in Animals 86
153A-8 Residue Analytical Methods 87
153A-9 Storage Stability Data 88
153A-10 Magnitude of the Residue in Plants 88
153A-11 Magnitude of the Residue in Animals 89
153A-12 Potable Water, Fish, and Irrigated Crop Studies 90
153A-13 Food Handling Establishment Studies 90
153A-14 Practical Methods for Removing Residues that
Exceed Any Proposed Tolerance (Section E
of a Petition) 90
153A-15 Proposed Tolerances (Section F of a Petition) 90
153A-16 Reasonable Grounds in Support of the Petition 90
Section G of a Petition
153A-17 Exemptions from the Requirement of a Tolerance 91
153A-18 Tolerances for Foreign Uses 91
153A-19 Rotational Crop Tolerances 91
153A-20 Tobacco Uses 91
153A-21 Data Requirements for Food Use vs. Nonfood Use 91
153A-22 Submittal of Analytical Reference Standards 91
153A-23 Special Considerations for Temporary
Tolerance Petitions 91
153A-24 Presentation of Residue Data 91
153A-25 Translation of Data 91
154A NONTARGET ORGANISM HAZARD GUIDELINES 92
154A-1 Overview 92
154A-2 Terrestrial Wildlife 96
154A-3 Aquatic Animals 102
154A-4 Nontarget Plant Testing 113
154A-5 Nontarget Insects 114
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iii
Tier I
154A-16 Avian oral 116
154A-17 Avian Respiratory Pathogenicity Test: Tier I 120
154A-18 Wild Mammal Toxicity and Pathogenicity Testing:
Tier I 124
154A-19 Freshwater Fish Toxicity and Pathogenicity
Testing: Tier I 127
154A-20 Freshwater Aquatic Invertebrate Toxicity
and Pathogenicity testing: Tier I 131
154A-21 Estuarine and Marine Animal Toxicity and
Pathogenicity Tests: Tier I 136
154A-22 Plant Studies: Tier I 142
154A-23 Nontarget Insect Testing for Toxicity/
Pathogenicity to Insect Predators and
Parasites: Tier I 147
154A-24 Honey Bee Toxicity/Pathogenicity Test: Tier I 152
Tier III
154A-25 Terrestrial Wildlife and Aquatic Organism
Toxicity Testing: Tier III 154
154A-26 Chronic Avian Pathogenicity and Reproduction
Test: Tier III 155
154A-27 Aquatic Invertebrate Range Testing: Tier III 159
154A-28 Fish life Cycle Studies: Tier III 161
154A-29 Aquatic Ecosystem Disruption Studies: Tier III 164
154A-30 Special Aquatic Tests - Tissue Culture,
Microorganism/Stress Interaction Tests 166
154A-31 Plant Studies: Tier III 167
154A-32 [Reserved] 167
Tier IV
154A-33 Simulated or Actual Field Testing for Mammals
and Birds: Tier IV 168
154A-34 Simulated or Actual Field Testing for Aquatic
Organisms: Tier IV 168
154A-35 Simulated or Actual Field Testing for Insect
Predators and Parasites: Tier IV 168
154A-36 Simulated or Actual Field Testing for Insect
Pollinators: Tier IV 168
154A-37 Simulated or Actual Field Testing for Plants:
Tier IV 168
155A TIER II ENVIRONMENTAL EXPRESSION DATA REQUIREMENTS FOR
MPCAS
155A-1 General Information 169
155A-2 Reporting and Evaluation of Data 174
155A-3 Through 155A-9 [Reserved] 175
155A-10 Tests to Determine Expression in a Terrestrial
Environment 175
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IV
155A-11 Tests to Determine Expression in a Freshwater
Environment 179
155A-12 Tests to Determine Expression in a Marine or
Estuarine Environment 182
156A PRODUCT PERFORMANCE GUIDELINES FOR MICRQBIAL PEST CONTROL
AGENTS
156A-1 Overview 184
156A-2 General Provisions 184
156A-3 Specific Provisions 186
157A EXPERIMENTAL USE PKKM1T GUIDELINES FOR MICROBIAL PEST
CONTROL AGENTS
157A-1 Overview 187
157A-2 Scope and Intent 187
157A-3 General Provisions 188
157A-4 Specific Data Requirements 189
158A LABEL DEVELOPMENT
158A-1 Product Label Requirements 192
APPENDIX
Data requirement tables 193
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Pesticide Assessment Guidelines
INTRODUCTION
I. Contents of Subdivision M.
Subdivision M provides testing and informational guidelines for
data to be submitted to support registration of Microbial (Part A)
and Biochemical (Part B) pest control agents. The following eight
section series and topics are covered:
151 Product analysis
152 Toxicology
153 Residue Hat-a
154 Nontarget organism hazards
155 Environmental fate and expression
156 Product performance
157 Experimental use permit data
158 Label development
The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)
establishes the Agency's authority over the distribution and use of
pesticide products. Before the Agency can register a pesticide,
FIFRA requires the Agency to have sufficient data to determine that
the pesticide, when used in accordance with widespread and commonly
recognized practice, will not cause (or significantly increase the
risk of) unreasonable adverse effects to humans or the environment
(Section 3 (c) (5) and (7) of FIFRA).
Part 158 of Title 40 of the Code of Federal Regulations (CFR),
specifies the kinds of data and information that must be submitted
to EPA to support the registration of each pesticide. This subdi-
vision provides detailed information relating to the data require-
ments listed in 40 CFR 158.690 and 158.740, including the conditions
under which each data requirement is applicable; the standards for
acceptable testing; the information that should be included in a
test report; guidance on evaluation and reporting of data; and
examples of protocols. In addition, scientific publications are
cited in the guidelines to provide useful information for designing
test protocols.
II. Scope of Subdivision M.
For regulatory purposes, the Agency has classified biological
and biologically derived pesticides into two major categories: the
microbial pest control agents i.e., microorganisms, and the bio-
chemical pest control agents e.g., pheromones, hormones, natural
insect and plant growth regulators, and enzymes. Biological and
biologically derived pesticides are typically naturally occurring,
specific to the target species, and, in the case of biochemicals,
typically have unique or non-toxic modes of action.
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Subdivision M
Because of these factors, the biological pesticides are roost appro-
priately characterized for health and environmental safety by
testing schemes which take their unique characteristics into ac-
count.
A. Microbial pest control agents (MPCAs). Pesticides referred
to as microbial pest control agents include, but are not limited
to, bacteria, algae, fungi, viruses, and protozoa as defined in
40 CFR 152.20. The guidelines apply to all microbial pest control
agents used as pesticides, including both those that are naturally
occurring, and those that are strain improved, either by natural
selection or by deliberate genetic manipulation.
These guidelines were developed for MPCAs in order to address
the special testing needs for these products. Unlike chemical
pesticides, MPCAs may survive and reproduce in the environment, and
may infect or cause disease in other living organisms. Thus, the
basic testing protocols are designed specifically to detect any of
these characteristics. Protocols for further testing emphasize
exposure or environmental expression in addition to expanded testing
of infectivity and pathogenicity.
B. Biochemical pest control agents (BPCAs).
Biochemical pest control agents generally fall into four biolo-
gically functional classes: semiochemicals, hormones, natural
plant regulators, natural insect growth regulators, and enzymes.
EPA has developed distinct data requirements for BPCAs in order to
facilitate the registration of this class of pesticides. BPCAs are
generally species specific and control their target pest by means
such as growth regulation or mating disruption. In contrast, con-
ventional pesticides are generally developed because they are toxic
(usually lethal) to a pest and less attention is given to the selec-
tivity of the pesticides for the target species. Furthermore, most
BPCAs are applied at very low rates, are highly volatile, or are
applied in bait, trap, or "encapsulated" formulations. Thus, the
application of most BPCAs results in less exposure to humans and
the environment than that from the use of most conventional pesticides.
Therefore, the likelihood of adverse effects from BPCAs will be
relatively lower than that for most conventional pesticides.
The regulations concerning data requirements for registration
(40 CFR 158) indicate at 158.65 that both biochemical and microbial
pesticides are generally distinguished from conventional pesticides
by their unique mode of action, low use volume, specificity to the
target species, or natural occurrence (as described above). The
regulation further states that, for biochemicals, the Agency will,
when necessary, evaluate products on an individual basis to determine
whether they are biochemical or conventional chemical pesticides.
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Pesticide Assessment Guidelines
The Agency has evaluated numerous candidate biochemicals on a
product by product basis. In each case, one of the major purposes
of this evaluation has been to determine whether, on the basis of
the above characteristics, the product can be sufficiently distin-
guished from conventional chemical pesticides to warrant regulation
as a biochemical product. Our experience has shown that no single
characteristic is sufficient to define a product as a biochemical.
Moreover, certain combinations of characteristics have been found
to be more important than others and thus, to date, products deter-
mined to be biochemicals have always possessed some combination of
the above characteristics. Experience has also shown that unique
mode of action and natural occurrence are two of the key character-
istics supporting the determination that a product is a biochemical.
III. Background of Subdivision M
microbial pesticide (Bacillus popilliae) was registered in 1948.
This pesticide was a naturally occurring bacterium. During the
late 1960s and early 1970s, interest in microbial pesticides began
to increase. As of 1987, there are 14 microbial pesticides used in
about 100 separate products registered for use in agriculture,
forestry, mosquito control, and homeowner situations.
In 1974, in recognition of the growing interest in, and concern
about microbial pesticides, the Agency began to sponsor a variety
of workshops, symposia, and panel discussions aimed at identifying
the relevant safety concerns for microbial pesticides. As early as
1978, at an EPA symposium titled "Viral Pesticides: Present Knowledge
and Potential Effect on Public and Environmental Health", the need
for sensitive identification and detection methods for inicroorganisins
as well as quality assurance provisions were clearly identified.
The Office of Pesticide Programs (OPP) issued a Policy Statement
on Biorational Pesticides which was published in the FEDERAL REGISTER
of May 14, 1979 (44 ER 23994). In it, OPP recognized microbial and
biochemical pesticides as distinct from conventional chemical pes-
ticides, and made the commitment to develop appropriate testing
guidelines. In 1979, OPP commissioned an American Institute of
Biological Sciences' expert panel to develop a "Human Hazard
Evaluation Scheme for Biorational Pesticides." The final report of
this expert panel formed the basis for the mammalian toxicology
unit of the testing guidelines for microbial pesticides.
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Subdivision M
OPP completed draft testing guidelines for Microbial and
Biochemical Pesticides in 1980. After review by the FIFRA
Scientific Advisory Panel (SAP) and public comment, these guidelines
were finalized in October, 1982, as Subdivision M of the Pesticide
Assessment Guidelines and published through the National Technical
Information Service (NTIS) in 1983 (EPA-540/9-82-028). The microbial
pesticide portion of the Subdivision M guidelines applies to both
naturally occurring and genetically modified pesticides. It was
also decided at that time that any additional data that would be
required for the registration of genetically modified microorganisms
would be determined on a case-by-case basis by EPA.
B. Registration requirements. The Data Requirements for
Pesticide Registration, 40 CFR Part 158, were published in the
FEDERAL REGISTER of October 24, 1984, (49 FR 42856). This regula-
tion contains data requirements for biochemical pesticides at 158.690
and for microbial pesticides at 158.740. These data requirements
were previously reviewed by the FIFRA SAP in October, 1983, as part
of Subdivision M of the Pesticide Assessment Guidelines.
C. Field testing r*yyTi">nements. The provisions for obtaining
Experimental Use Permits (EUP) for testing pesticides is codified
in 40 CFR Part 172. This regulation includes a presumption that an
EUP will not be necessary for small-scale field testing (non-food
uses on not more than ten acres of land or not more than one surface-
acre of water, providing that the water not be used for irrigation
purposes, drinking water supplies, or body-contact recreational
activities). Such a presumption is appropriate for chemical pesti-
cides which have no independent mobility or reproductive capability
and, therefore, when applied in small scale field studies generally
have very limited potential for causing adverse effects outside the
treated area. Similarly, when used in small scale field tests,
naturally-occurring microbial pesticides are subject to natural
control or dissipation mechanisms. However, genetically altered
microbial pesticides may not be subject to natural control or dis-
sipation mechanisms and thus may be capable of spreading beyond the
site of application with the potential for causing adverse effects.
Therefore, small scale field studies with these types of microbial
pesticides could raise many of the same concerns as more extensive
use of conventional pesticides, and the presumption that an EUP is
not required may not be appropriate for these pesticides.
To address these issues, the Agency developed an interim
policy requiring notification under FIFRA prior to small scale
field testing of genetically altered and nonindigenous microbial
pesticides in order to determine the need for EUPs prior to release of
these MPCAs into the environment. This interim policy was published
in the FEDERAL REGISTER of October 17, 1984 (49 FR 40659), and also as
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Pesticide Assessment Guidelines
part of the Proposal for a Coordinated Framework for Regulation of
Biotechnology in the FEDERAL REGISTER of December 31, 1984 (49 FR
50856). This policy was revised in accordance with public comments
and was re-published in the FEDERAL REGISTER of June 26, 1986, as
part of the Coordinated Framework for Regulation of Biotechnology;
Araiauncement. of Policy and Notice for Public Comment (51 FR 23302).
The Agency is currently in the process of promulgating amendments
to 40 CFR Part 172 to implement this policy.
IV. Revisions to Subdivision M.
The Agency has gained considerable experience in the risk
assessment of MPCAs and BPCAs since Subdivision M was published in
1983. Accordingly, the need for revising and updating portions
of the guidelines, particularly the sections dealing with micro-
organisms, has became apparent. This revision is intended to better
address the needs of both testing laboratories and OPP scientific
staff. The Agency recognizes that further revisions will be needed
as we learn more about the behavior of microorganisms in the envi-
ronment. Accordingly, OPP is actively sponsoring research in this
area through the EPA Office of Research and Development.
A key editorial feature of these revised guidelines is that the
BPCA and MPCA sections have been separated into two distinct parts
of the document. The revised version also reflects an extensive
updating of testing guidelines for MPCAs.
In addition, the immunotoxicology section for testing BPCAs
has been upgraded from the original version. These iamtunotoxicology
guidelines were presented to the FIERA SAP for review on March 24,
1987, and their comments have been incorporated. No other changes
have been made in the BPCA guidelines than the editorial changes
necessary in separating them from the MPCA guidelines.
The revised guidelines utilize the tier testing scheme set
forth in 1983 with the original publication of Subdivision M.
Three of the major section series, Toxicology, Nontarget Organism
Hazard, and Environmental Fate and Expression, use the tiered testing
scheme (see Appendix) to ensure, to the best extent possible, that
only the minimum data sufficient to make scientifically sound
regulatory decisions will be required. The Agency expects most of
the MPCAs and BPCAs will require testing only in the first Tier.
Moverover, the Agency believes that the Tier I test requirements
represent a reasonable approach to evaluating risk related to the
use of pesticides, and is one in which negative results would allow
a high degree of confidence in the safety of the test agents.
V. Relationship to Other Subdivisions of the Guidelines.
This subdivision describes a complete set of requirements
for biochemical and microbial pesticides. In addition to tests that
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Subdivision M
are unique to these classes of pesticides, some tests are identical
to those used for conventional pesticides. In these cases, references
are made to other subdivisions. Each section series in this sub-
division corresponds to a subpart of the guidelines for conventional
chemical pesticides.
To illustrate:
Series 151 Product Analysis corresponds to Subdivision D - Product
Chemistry;
Series 152 Toxicology corresponds to Subdivision F - Hazard
Evaluation: Humans and Domestic Animals;
Series 153 Residue Analysis corresponds to Subdivision 0 - Residue
Chemistry;
Series 154 Nontarget Organism Hazard corresponds to a combination
of Subdivision E - Hazard Evaluation: Wildlife and Aquatic
Organisms, Subdivision J - Hazard Evaluation: Nontarget Plants,
and Subdivision L - Hazard Evaluation: Nontarget Insects;
Series 155 Environmental Fate and Expression corresponds to
Subdivision N - Environmental Fate;
Series 156 Product Performance corresponds to Subdivision G -
Product Performance;
Series 157 Experimental Use Guidelines corresponds to Subdivision
I - Experimental Use Permits; and
Series 158 Labeling Development corresponds to Subdivision H -
Labeling for Pesticides and Devices.
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Pesticide Assessment Guidelines
A. MICRQBIAL PEST CONTROL AGENTS
150A GENERAL TNPOPMATION
15QA-1 Overview.
(a) Scape and purpose. This subdivision describes the kinds
of data required by 40 CFR Part 158 to support the field testing
and registration of naturally occurring or genetically altered
microbial pest control agents (MPCAs). Each section in this sub-
division specifies the kinds of data required by Part 158, the
standards that the studies must meet, and the conditions under
which each study is required as specified in Part 158.
(b) Data requirements for registration. MPCA data requirements
are listed in 40 CFR 158.740. Testing is required in the areas of
Product analysis, Toxicology, Residue analysis on food crops, and
Ecological effects and Environmental expression. These guidelines
contain revised data requirements as listed in the Appendix to this
document.
Product analysis requirements include the necessary data and
information to identify the active ingredient and any inert substances
that have been added, and to guard against chemical and biological
contamination both prior to registration and during production of
the MPCA. This information is required for all MPCAs.
Toxicology requirements are set forth in three tiers. Tier I
consists of a battery of short term tests designed to evaluate
potential for toxicity, infectivity, and pathogenicity- Tier II is
designed to evaluate the particular situation when, in the absence
of evidence of pathogenicity, either toxicity or infectivity is
observed in Tier I. Tier III contains tests that may resolve
issues of known or suspected human pathogenicity and tests for
particular adverse effects of intracellular parasites of mammalian
cells.
Residue data describe the quantity of MPCA or its associated
toxins that might appear on food or feed crops. These data are
required only if there are significant human health concerns arising
from the toxicology testing.
Ecological effects and environmental expression testing also
have been placed into tiers. Tier I consists of maximum dose
single species hazard testing on nontarget organisms. If adverse
effects are observed in Tier I, then the potential exposure to the
MPCA is estimated by means of Tier II testing for population dynamics
(fate and expression) in the environment. If Tier II tests show
that there may be significant exposure to the MPCA, then Tier III
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Subdivision M
studies to determine a dose-response effect or to examine certain
chronic effects will be performed to determine if the minimum
infective dose is less than the exposure or if there are other
considerations that would decrease the observed effects in the
environment. Tier IV tests, under simulated or actual environmental
conditions, are to be designed on a case-by-case basis to evaluate
any specific problem that can not be resolved by lower tier testing.
(c) Data requirements for field testing.
At the date of these Guidelines, the Agency is in the process
of promulgating amendments to 40 CFR Part 172 to implement the
policy published in the June 26, 1986 FEDERAL REGISTER (51 FR 23302)
for submission of notifications prior to conducting small scale
field tests (testing involving 10 acres or less of land or 1 acre
or less of water) of certain microbial pesticides (see section III
C., above). In the interim, anyone considering small scale field
tests of microorganisms covered by the policy should consult with
the Agency.
Pursuant to Section 5 of FIFRA and 40 CFR 172.2, the Agency
provides for issuance of Experimental Use Permits for large scale
field testing of pesticides, including all MPCAs, involving
terrestrial application of more than 10 acres or direct application
to more than 1 acre of water. The Agency encourages applicants for
EUPs to consult with OPP scientists to develop an appropriate
testing scheme.
The data required prior to field testing of MPCAs may consist
of any, or all, of the tests required for registration since these
living microorganisms have the potential to multiply to high exposure
levels, depending on the their ability to survive, reproduce and
compete for dominance in the environment. However, the Agency
recognizes the need to limit testing in the course of development
of pesticides and intends to make every effort to restrict field
testing data requirements to only those necessary to evaluate that
particular field test.
Reduced data requirements for EUPs may be justified on a case-by-
case basis by consideration of certain exposure factors (e.g.,
limited capacity for the MPCA to survive at, or disseminate from,
the field test site, and containment or mitigation provisions in
the test protocols). Human health and ecological effects testing
will be limited to the most likely areas of concern as predicted by
a careful consideration of the known properties of the MPCA and
similar microorganisms. As much information as possible should be
submitted to the Agency to allow for analysis of the potential
risks of the field tests. Specific information recommendations for
this preliminary assessment of both Notifications and Experimental
Use Permit Applications are discussed in 157A of these guidelines.
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Pesticide Assessment Guidelines
(d) Principles for determining data needed for field tests.
•Hie full battery of tests for registration of MPCAs was designed
to give basic hazard and exposure information for a microorganism
with totally unknown properties. In actual practice, an MPCA is
usually well identified which may facilitate prediction of the
properties and behavior of the MPCA. This is particularly true for
the areas of human health and plant pathogenicity. Clinical medicine
and agricultural science have identified most microorganisms asso-
ciated with diseases. If the MPCA is taxonomically similar to a
clinically or agriculturally significant microorganism, this par-
ticular area of concern should be examined closely, possibly, as
provided in 40 CFR 158.75, by requiring additional testing beyond
that specified in 40 CFR 158.740. Conversely, if the MPCA belongs
to a group of microorganisms that have never been found in asso-
ciation with any disease, a case may be made for reducing, or
waiving, the testing requirements for this area of concern.
Part 158 of 40 CFR contains provisions for granting waivers for
data requirements in response to specific written requests by ap-
plicants (40 CFR 158.45). OPP encourages applicants to discuss
their preliminary testing plans with OPP scientists. Waivers of
some testing requirements may be appropriate for certain MPCAs.
Ihis tailoring of the testing battery on a case-by-case basis relies
on both an accurate description of the MPCA and the existence of a
reliable taxonomy for the class of microorganism to which it belongs.
Some microorganisms have been more closely examined than others and
have a larger data base from which to draw conclusions. In addition,
certain kinds of microorganisms are more amenable to classification
than others. In general, human and plant pathogenic bacteria have
been best classified due to their health and economic significance.
Other microorganisms, particularly protozoa and fungi, might not be
as well studied or described, and it may be difficult to reliably
predict their properties from a taxonomic description. In this
case, it may be more difficult to justify waiving test requirements.
An additional factor in determining the extent of testing
that may be necessary for risk assessment is the degree of species
specificity shown by the MPCA. Ihis is of primary importance in
assessing ecological risk. Most MPCAs are designed to produce
adverse effects against a target species. Careful scientific con-
sideration on a case-by-case basis must be given to the selection
of nontarget species to be tested (e.g., beneficial insects, envi-
ronmentally or commercially significant plants, or wildlife) so as
to include species that are most likely to be susceptible.
Because of the difficulty in providing definitive exposure pre-
dictions from currently available ecological methods, the Agency takes
the approach that hazard testing on nontarget organisms should be
submitted initially (Tier I tests). If significant adverse effects
are identified in Tier I tests, then ecological exposure tests
(Tier II) are performed to attempt to quantify levels of the MPCA
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10
Subdivision M
to which the susceptible nontarget species may be exposed. Although
normally requested at a Tier II level, definitive ecological exposure
data showing that the MPCA will not survive or persist in the envi-
ronment would be good support for a request for waiver of some or
all of Tier I testing requirements.
150A-2 Definitions.
(a) Terms used in this subpart are defined in FIFRA,
in 40 CFR 162.3, and in the following sections of the guidelines;
60-2 of Subdivision D
70-2 of Subdivision E
80-2 of Subdivision F
90-2 of Subdivision G
100-2 of Subdivision H
110-2 of Subdivision I
120-2 of Subdivision J
140-2 of Subdivision L
160-2 of Subdivision N
(b) In addition, for the purposes of this subdivision:
(1) "Animal" means all vertebrate and invertebrate species,
including, but not limited to humans and other mammals, birds, fish,
and shellfish.
(2) "Aquatic animals" means all vertebrates and
invertebrates that inhabit fresh, estuarine, or marine waters
for all or part of their life cycles.
(3) "Aquatic use" means the use of a pesticide in a
fresh water, estuarine, or marine aquatic system by either
direct application or direct discharge of treated water.
(4) "Biological control agent" means a living organism
introduced into the environment to control the population or
biological activities of another life form considered to be
a pest under sec. 2(t) of FIFRA.
(5) "Dose" means a quantity of material, whether liv-
ing or not, to be applied to an animal at one time.
(6) "Dosing regimen" means a systematic schedule of
doses.
(7) "End-use Product (EP)" is an MPCA-containing product that is
registered or intended for direct use or application for pest control
purposes. In some cases, an EP is identical to the manufacturing-use
product (MP) [the technical grade of the active ingredient (TGAI) or
formulation intermediate (FI) ]. In other cases, an EP is formulated
from the MP by addition of inert ingredients such as antioxidants
or other stabilizers, suspending agents, carriers, encapsulating
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Pesticide Assessment Guidelines
materials, wetting agents, or anticaking compounds. The inten-
tionally-added inerts may influence MPCA storage stability/ viability
as well as deposition and persistence at the end-use site. In some
cases, an EP is manufactured via an integrated formulation process,
i.e., the MP used for formulation is not a separate registered
product.
(8) "Environmental expression" means the extent and
manner in which a microorganism establishes and maintains
its presence in an ecological habitat.
(9) "Estimated environmental concentration" means an
estimate of the concentration of a MPCA occurring in or on various
media (i.e., soil, water, air) after pesticide application, as
determined from the results of environmental fate or expression
Tier II testing.
(10) "ID 50" means the amount of material required to
produce overt disease symptoms in 50 percent of the test
animals.
(11) "Infectivity" is the ability of a microorganism to cross
or evade natural host barriers to infection.
(12) "Maximum expected environmental concentration"
means the highest concentration of a pesticide occurring at
any given time (usually immediately after application) at a
site or in a medium (e.g., water, vegetation, or soil) as
determined from the pesticide application rate.
(13) "Manufacturing-use Product (MP)" is a preparation containing
the MPCA in question that is used to formulate an end-use product
(EP; see above). An MP either is the technical grade of the active
ingredient (TGAI) or a formulation intermediate (FT). An FI is a
product containing the TGAI to which other ingredients deliberately
have been added (e.g. stabilizers, dispersants, diluents).
(14) '"Maximum hazard testing" means a testing scheme
that is designed to maximize any toxic or pathogenic effects
of the test substance on the test (non-target) organism.
(15) "Microbial pest control agent" means any of those
microorganisms including (but not limited to) bacteria, fungi,
viruses, and protozoa as defined in 40 CFR 162 that are used to
control pests.
(16) "Morbidity" means the evident state of disease.
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Subdivision M
(17) "Moribund" means approaching death.
(18) "Mortality" means the state of an animal or plant
in which all vital functions have ceased.
(19) "Natural occurrence" means the presence of an
organism in its normal habitat where it grows, develops, and
reproduces.
(20) "Pathogenicity" is the ability to inflict injury and
damage in the host after infection, and depends on host resistance
or susceptibility.
(21) "Plant" means any member of the Kingdom Planta.
(22) "Pure Active Form of Each Ingredient (PAI)" is a preparation
containing pesticidally functional units of the MPCA in question
obtained after the application of the most rigorous purification
procedures. Where techniques are used to genetically alter the
MPCA, the genetically altered strain is considered as the basis for
defining the pure active form of the MPCA. The purest form of the
MPCA that can be obtained is a preparation that is free of any
other biological forms and free of contaminating growth media or
host substrate material. Chemical pesticidal product from genes
that have been engineered into a microorganism also may be considered
as separate active ingredients.
(23) "Purest infective form" means that preparation of
infective virus containing the least amount of extraneous
material.
(24) "Technical Grade of the Active Ingredient (TGAI)" is a
material containing the microbial pest control agent (MPCA) in
question which is produced commercially, or in a manner equivalent
to the planned commercial process, and to which no ingredient
intentionally has been added except for purposes of MPCA growth or
replication, or typical purification. The TGAI is considered to be
the purest preparation resulting from a typical production process,
and is the preparation intended for distribution and/or formulation
into a formulation intermediate (FI) or end-use product (EP).
Where techniques are used to genetically alter the MPCA, the gene-
tically altered strain is considered as the basis for defining the
technical grade of the MPCA. Each pesticidal product from genes
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Pesticide Assessment Guidelines
that have been engineered into a microorganism also is considered
as an active ingredient. The technical grade of each pesticidal
product from introduced genes is that form which exists along with
the technical grade of the MPCA in question after a typical production
process.
(25) "Terrestrial wildlife " means non-domestic birds or
animals.
(26) "Toxicity" is the injury or damage in a host caused
by a poison or toxin where infection by and/or replication
or viability of the microorganism are not necessarily required.
(27) "Toxin" means a poisonous substance, generated by
a microorganism, plant, or animal, capable of causing injury
or damage when it interacts with host cells.
(28) "Typical end-use product" means a pesticide product
representative of a major formulation category (e.g.,
emulsifiable concentrate, granular product, wettable powder)
that contains the active ingredient of the registration
applicant's product.
(29) "Virulence factors" mean the traits of a microorganism
that allow for pathogenicity.
150A-3 General provisions.
(a) Scope. The standards contained in this section ap-
ply to all studies in this subdivision unless modified for use in
a specific section.
(b) Basic standards for testing.
(1) Test substance for biological and environmental studies.
It is advised that appropriate representatives of the EPA be consul-
ted prior to testing in order to determine the form/purity of the
MPCA that should be tested to support the registration of each MP
and each EP. It is recognized that certain forms of the MPCA may
be inappropriate for certain tests. In general, the form (e.g.
vegetative cell, spore, cyst, virion) of the microorganism to be
tested should be equivalent to the form that is intended for regis-
tration. The test microorganism also should be equivalent to that
intended for registration with respect to stage of growth, pos-
session of organelles and appendages, and expression of phenotypic
traits (including products from genes that have been intentionally
introduced into the microorganism). If significant exposure to
other forms of the microorganism is expected, or if changes in
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Subdivision M
form of the microorganism occur, or are expected to occur in target
or non-target species, then these forms also may have to be tested.
In general, the following principles should be followed:
(i) Tests requiring use of the technical grade of the active
ingredient shall be conducted with the manufacturing-use product if
the TGAI and MP are identical, or with the technical grade of the
active ingredient used to produce the manufacturing-use or end-use
formulated pesticide product if not identical.
(ii) The lot of the substance tested should be the same
throughout the duration of the study, and the test sample should be
stored under conditions that maintain purity and stability. If the
stability of the test substance cannot be maintained for the duration
of the study or if, for other reasons, it is not possible to use
the same lot throughout the test, subsequent lots of the test substance
shall be selected to be as nearly identical to the original lot as
practical. Chemical or biological assays shall be performed to
ensure composition identity and consistency.
(iii) Each lot of the test substance shall be analyzed, to
the limits of technical feasibility/ and the name and quantities of
ingredients, contaminants, and impurities listed. The determination
shall include the quantity of unknown material, if any, so that
100% of the test sample is accounted for. The test substance shall
be within the limits of purity, if any, certified in accordance
with 151A-15 of this subdivision.
(iv) If the test or control substance is to be incorporated
into feed or other vehicle, the period during which the test or
control substance is stable or viable in such a mixture should be
determined prior to the start of the study. No mixture of test or
control substance with the feed or vehicle shall be maintained or
used during a period exceeding the known stability or viability of
the test or control substance in the mixture. Alternatively,
determinations of the stability or viability of the test or control
substance in random samples of the diet or vehicle mixture shall be
made at least monthly during the study to ensure that proper mixing,
formulation, and storage procedures are being followed and that the
appropriate concentration of the test or control substance is con-
tained in the mixture.
(v) If the test or control substance is incorporated into
feed or other vehicle, its homogeneity and concentration in the
diet shall be determined prior to the start of the study and each
time a new mixture is prepared. Random samples of the mixture
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Pesticide Assessment Guidelines
shall be analyzed at least monthly to ensure that proper mixing,
formulation, and storage procedures are being followed, and that
the appropriate concentration of the test or control substance is
contained in the mixture.
(vi) In addition to or in lieu of data otherwise required by
this subdivision, the Agency may require, after consultation with
the applicant, data derived from testing to be conducted with:
(A) an analytically or microbiologically [e.g., purest
infective form (PIF) for viruses] pure grade of an active ingredient;
(B) the labile form of infectious material (e.g., non-
occluded virus);
(C) an inert ingredient of a pesticide formulation;
(D) a contaminant or impurity;
(E) a metabolite (from animals or plants) or degradation
product of an active or inert ingredient;
(F) the end-use formulated product;
(G) any additional substance (including other pesticides
recommended for tank-mixing with the test substance) that
enhances the virulence or toxicity of the product for which
registration is sought; or
(H) any combination of the substances mentioned in paragraphs
(b) (1) (vi) (A) through (H) of this section.
(2) Administration or application of test substance and
vehicles.
(i) The manner of administration or application of the test
and control substance for biological or environmental testing shall
be selected so as to maintain accuracy of the dosage or treatment.
(ii) A vehicle other than water or saline, used to
dissolve or dilute the test substance or positive control
substance shall be chosen to possess the following character-
istics if possible:
(A) It does not alter the absorption, distribution,
metabolism, or retention of the test substance;
(B) It does not alter the chemical or biological proper-
ties of the test substance or enhance, reduce, or alter the
pathogenic or toxic characteristics of the test substance;
(C) At the levels used in the study, it does not produce
physiological effects and is nontoxic; and
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Subdivision M
(D) It should be identical to, or closely resemble
the vehicle, if any, used in the pesticide product. It
should be identical to the vehicle if possible.
(3) Controls for biological and environmental studies.
Controls are used in biological or environmental studies
required by this subdivision to ensure that observed effects are
associated with the test substance exposure. The appropriate
control groups shall be identical in every respect to the
treated groups except for exposure to the test substance.
In studies involving animals or plants, all controls shall,
to the extent possible, be from the same source, be of the
same age, receive the same care, and receive the same nutri-
ents as the animals or plants receiving the test substance.
To prevent bias, a method to randomly assign organisms
to treatment and control groups is required and must be
referenced in the report.
(i) Untreated (negative) controls. Untreated (negative)
control groups are usually required. Untreated controls re-
ceive neither the test substance nor any ancillary material
(vehicle).
(ii) Controls treated with inactivated MPCAs. In
certain circumstances, deleterious effects may be produced in
test animals through a mechanism other than active infection
(e.g. anaphylaxis). This control group may provide information
useful in determining the mechanism of pathogenesis.
(iii) Vehicle control groups. (A) If a vehicle, other than
water or saline, is used to administer the test substance, a
concurrent vehicle control group may be required. Vehicle control
groups receive treatment with the vehicle alone, and the vehicle is
usually administered at the highest level that the vehicle is
administered in any test group in the study. Consult individual
sections of this subdivision for those tests where a vehicle control
is required or recommended.
(B) As provided in paragraph (b) (3) (iii) (A) of this section,
the vehicle should be selected on the basis of information estab-
lishing that it is non-toxic at the levels used in the study, has
no independent physiological effects, and does not alter the chemistry,
pathogenicity, or toxicity of the test substance. If, however,
there are insufficient data on the effects of the vehicle, testing
of the vehicle is required.
(iv) Positive controls. Positive controls generally are not
required. These serve as internal quality controls, and demonstrate
known test organism sensitivity and response to known toxic or
infective agents. They are also used to ascertain if a strain or
species reacts similarly to another strain or species when exposed
to the same known or standard toxicant or infective agent. Consult
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Pesticide Assessment Guidelines
individual sections of this subdivision for those tests where a
positive control is required or recoiranended.
(iv) Historical controls. Historical control data are
required when the Agency desires information on longevity,
spontaneous diseases, or other characteristics of a species
or strain selected for study, and for certain comparative or
statistical purposes. Consult individual sections of this
subdivision for those tests where historical control data are
required.
(v) Additional controls may be required as dictated by
test design.
(c) Special test requirements. In addition to the data
required in this subdivision, data derived from other tests may,
under unusual circumstances, be required by the Agency in
order to make judgments regarding safety to humans, domestic
animals, and other nontarget organisms. Such data will be
required where special problems are encountered. Test methods
will usually be derived from tests already described or cited
in other subdivisions of these guidelines. Such data requests
may relate to a proposed pattern of use, a toxicological
mode of action, or a unique chemical or microbial property.
The data requested will be specific to the problem. Examples
of test requirements for unusual circumstances include but
are not limited to: certain chemical property data from Sub-
division D ( 61-7), and certain toxicity data from Subdivision F.
150A-4 Reporting of data.
Each test report submitted under this subdivision shall
satisfy the reporting requirements of this section, unless
a specific section elsewhere in this subdivision directs
otherwise.
(a) General requirements.
(1) Identification. Each test shall identify:
(i) The name and address of the laboratory or site where
the test was performed and
(ii) The party (s) primarily responsible for any written
or other matter contained in the report, and the portions of
the report for which each party is responsible.
(2) Verification. Each test report shall be:
(i) Signed by each of the senior scientific personnel,
including the laboratory director responsible for performing
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Subdivision M
and supervising the testing and preparing, reviewing, and
approving the test report, and;
(ii) Certified by the applicant or an authorized agent
of the applicant as a complete and unaltered copy of the
report provided by the testing laboratory, whether independent
or owned, operated, or controlled by the applicant.
(b) Format and content. The test report shall include
all information necessary to provide a complete and accurate
description and evaluation of the test procedures and results.
Ihe test report shall contain at least four parts: a summary
and evaluation of the test results, a description of the
test procedures, a listing of the data and information
required by each applicable section of this subdivision, and
a section in which data and findings are discussed. Metric
units of measurement must be used although English units may
be included where appropriate. The systems may not be mixed
(e.g., mg/qt.).
(1) Summary of test results. This section of the
test report shall contain a summary of the data and significant
findings.
(2) Description of the test procedure. This section of
the test report shall contain a full description of the test
procedure. If an applicant believes any of the reporting
requirements are not applicable, he shall submit an explanatory
statement to this effect. A full description of the test
procedure shall include but not be limited to:
(i) Deviation from standards. The report shall indicate
all ways in which the test procedure fails to meet applicable
standards for acceptable testing contained in this subdivision,
and shall state the reasons for such deviations.
(ii) Test methods. Specification of test methods,
including a full description of the experimental design
and procedures, the length of the study, and the dates on
which the study began and ended, shall be stated.
(iii) Substance tested. Identification of the test
substance shall be provided, including:
(A) If the test substance is microbiological: scientific
name and, to the extent possible, serotype and strain or
other appropriate designated type, and, to the extent possible,
a qualitative and quantitative determination of composition
(including names and quantities of known contaminants and
impurities, within technically feasible limits). The determ-
ination shall also include quantities of unknown materials,
if any, to account for 100% of the sample;
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Pesticide Assessment Guidelines
(B) Manufacturer and lot number of the test substance, and
relevant properties of the substance tested, (i.e., physical state,
pH, stability, and purity) ; and
(C) Identification and composition of any vehicles or other
materials (e.g., diluents, suspending agents, emulsifiers, virulence
enhancers) used in administering the test substance.
(iv) An i Trial and plant dat^u Animal and plant data shall
include:
(A) Species and strain used and reasons for selection of
species (if the species is other than the species recommended
or required by sections of this subdivision) .
(B) Source of supply of test organisms;
(C) Disease history of the test animals;
(D) Description of any pre-test conditioning;
(E) Method used to randomly assign animals or plants
to test or control groups;
(F) Numbers of animals of each sex. in each test or control
group; and
(G) Age and condition of animals or plants at beginning of
study.
(v) Environmental conditions. A description of the
environmental conditions under which the testing was conducted
shall be reported. Further details may be provided by spe-
cific testing sections elsewhere in this subdivision.
(vi) Treatments or doses. For studies where test substance
applications, treatments, or dosings are made, a complete description
of such shall be reported. Further details may be provided by
specific testing sections elsewhere in this subdivision.
(vii) Treatment for dis^apes not ranged bv the test
substance. Test animals or plants with a history of disease shall
not be used for microbial pesticide testing. Ihe feed must be
antibiotic free. For MPCAs where test organisms have been treated
with some agent or manipulated by some system to prevent or control
infectious diseases not caused by the test substance, a full description
of such treatment or manipulation must be reported. Such description
should include:
(A) Identification of the test organisms affected and
the disease organism involved;
(B) The nature and severity of the disease;
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Subdivision M
(C) The date of onset and duration of the disease;
(D) The nature of the treatment or manipulation used to
control or eliminate the disease, and the dates of such
actions; and
(E) The outcome of the treatments in relation to the
disease and to the test results.
(viii) Observations. Method, frequency, and duration of
observations made during the study shall be reported. Other related
specific information to be reported may be provided by specific
testing sections elsewhere in this subdivision.
(ix) Availability of raw data, specimens, and samples of the
test substances. The location of all raw data, specimens and
samples of the test substances which are retained in accordance
with 40-5 and 151A-11, and the name and address of the individual
who is responsible for the archives and the name and address of the
recognized culture collection, shall be reported.
(x) References. References must be provided for the statistical
and other methods employed for analyzing the data, and for any
published literature used in developing the test protocol, performing
the testing, making and interpreting the observations, and compiling
and evaluating the results.
(3) Reporting the results and evaluation of specific
tests. The test results and any evaluations of test results should
be reported in accordance with the requirements of the individual
specific testing sections of this subdivision. Such results and
evaluations include all data, information, and analysis necessary
to support the registration application and its corresponding product
label claims, directions, and precautions. The report must be at
such detail that a reviewing scientist has sufficient information
to reach an independent conclusion from the data.
(4) Discussion section. This section of the test report will
contain a full scientific discussion of any and all positive or
unexpected negative results and findings. All aberrant data shall
be noted and explanations, based on sound scientific principles,
given. Any conclusions arrived at by the study author(s) should be
included.
(c) Statistical procedures. (1) General. Appropriate
statistical methods shall be used to summarize experimental data,
to express trends, and to evaluate the significance of differences
in data obtained from different test groups. The methods used
shall reflect the current state of the art.
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Pesticide Assessment Guidelines
(2) Standard deviation and standard error. All data averages
or means shall be accompanied by standard deviations, to indicate
the amount of variability in the data. In addition, the standard
errors of the means should also be calculated, as they are useful
in comparing means from different test groups; however, notations
of statistically significant differences accompanied by the confidence
level or probability should also be used in place of standard error
determinations. Other methods of expressing data dispersion may
also be used, when appropriate.
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Subdivision M
Series 151A: PRODUCT ANALYSIS GUIDELINES FOR MICROBIAL
PEST CONTROL AGENTS
The product analysis data requirements for microbial pest
control agents under 151A-10 through -16 of this subdivision, to
some extent, parallel those for conventional chemical pesticides as
specified in Subdivision D. However, due to the unique nature,
composition, and mode of action of the MPCAs, there are some important
differences. For example, protozoa, bacteria, fungi, and viruses
should be identified to the extent possible by taxonomic position,
serotype, composition, and strain, or by any other appropriate
specific means. This information would take the place of chemical
name and structural formula information for conventional pesticides.
As a result, the guidelines in 151A-10 through -16 generally
reference the corresponding section in Subdivision D and indicate
those portions of Subdivision D which do not apply.
In addition, the Agency must be reasonably assured that the
methods used and the data submitted are capable of demonstrating
that the microbial pesticide used in the field is the same as
that which was tested for safety.
151A-1 Overview
(a) Scope of product analysis requirements. This
section series outlines guidelines for the submittal of data and
information on product analysis in support of applications for
microbial pest control agents (MPCA's). These guidelines generally
parallel those for conventional chemical pesticide products
specified in Subdivision D.
151A-2 through 9 [Reserved]
151A-10 Product Identity and Disclosure of Ingredients
(a) Product identity. As required by 40 CFR 158.740, each
application for registration of an MPCA shall contain the following
information: the product name; the trade name(s) (if different).
The company code number(s) may be given.
(b) Confidential Statement of Formula. As required by 40 CFR
158.740, an application for registration of a product shall
contain a Confidential Statement of Formula. This statement shall
include the nature and quantity of the active ingredient (s) and
diluents and the identity and purpose of inert ingredients such
as ultraviolet screens, stickers, spreaders, and other such
material. The appropriate EPA form shall be used (i.e., Form
8570-4). The name of each ingredient in the product for which
62-2 of Subdivision D requires certified limits to be established
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Pesticide Assessment Guidelines
shall be listed. A separate Confidential Statement of Formula is
required for each alternate formula of a product. See Section
10 of the Act for requirements related to the protection of
trade secrets.
(c) Information on ingredients. Information on ingredients
is required by 40 CFR 158.740 to support each application for
registration. (1) The identification of the MPCA(s) in the product
shall (to the extent possible) include the following:
(i) The taxonomic position, serotype, strain, or any other
appropriate designation. The precise test procedures and criteria
used for identification [i.e., the morphological, biochemical,
analytical (physical, chemical), serological, or other identification
means] and the results of such tests should be provided. If the MPCA
contains plasmids or other extrachromosomal genetic elements involved
in pesticidal activity/ pathogenicity, toxicity, etc., these must
be identified as well as any known phenotypic characters coded by
such elements and their stability. Whether or not a wild type or
genetically altered strain is considered to be a new active ingredient
will be determined on a case by case basis upon comparison with
existing related strains;
(ii) The common, alternative, and superseded names;
(iii) The natural occurrence of the organism, its relationship
to other species (particularly those that are pathogenic), and
its history;
(iv) A description of the morphological types of the MCPA and
any unusual morphological, biochemical, or resistance characteristic(s)
of the organism if such characteristic (s) are different from the
classic description of the organism;
(v) The amount of MPCA present in the product in recognized
units of potency, percentage of weight, units of MCPA per unit
weight or volume of product, or other appropriate expression of
biological activity.
(vi) The biological properties of the active agent with
respect to target species, pest host range, life cycle, and mode of
action. With respect to the properties of the microbial agent, any
known or potential hazard (such as inf activity) to mammals (including
humans), the environment, and nontarget species should be discussed.
(vii) If the MPCA in question has been genetically altered.
then, in addition to (c) (1) (i-vi), the methods used to genetically
alter the microbe should be provided. In the case of genetically
altered products, the identity of the inserted or deleted genetic
material (source, nature, size, base sequence data and/or restriction
endonuclease map), information on the gene control region, descriptions
of the phenotypic traits to be gained or lost, and information as
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Subdivision M
to the genetic stability (reversion tendency or rate of exchange/
transfer with other organisms) of the genetically altered chromosomal
region or extxadhromosomal entity are to be discussed. Genetic
material adjacent to the intentionally inserted gene(s) which may
have been engineered into the recipient are to be fully character-
ized and the likelihood of expression must be provided.
(2) An application for registration shall contain the
following information on each ingredient, other than the MPCA(s),
listed in the Confidential Statement of Formula required in
paragraph (b) of this section which is known to be present or
which might reasonably be identified in the pesticide product.
(i) Percentage composition (by weight) of each ingredient;
the number of units per unit volume or weight is needed for
microbial impurities; viability data in terms of PFU, CPU, etc.
per unit weight or volume of product;
(ii) Whether the ingredient is an active ingredient, an
intentionally added ingredient, or an impurity;
(iii) Ihe chemical name from the Chemical Abstracts 1972-
1976 Index of Nomenclature, or other well-defined name. If one
or more impurities are microbial agents, then the agents are to
be classified/identified according to acceptable nomenclatural
systems;
(iv) Hie Chemical Abstracts (CAS) Registry Number;
(v) Ihe product name, the trade name, and the common name;
(vi) Ihe experimental or internal code number;
(vii) For each active ingredient other than the MPCA, the
empirical formula, and the molecular weight or the molecular
weight range;
(viii) Ihe structural formula (when known); and
(ix) Ihe composition limits for each ingredient for which
62-2 of Subdivision D requires limits to be certified. This
information is to be listed on the Confidential Statement of
Formula, EPA Form 8570-4 revised 2/85 (or superseding revisions).
151A-11 Manufacturing Process
As required by 40 CFR 158.740, each application for
registration of a manufacturing-use product or end-use product
shall contain a description of the basic manufacturing process
as required in 61-2 of Subdivision D. Ihe starting and inter-
mediate materials should be listed (including EPA Registration
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Pesticide Assessment Guidelines
No. and/or source and purity) together with the steps taken to
ensure the integrity of these materials, and the steps taken to
limit the extraneous contamination, both chemical and biological,
in preparations of the unformulated MPCA. This description shall
include the procedures used by the manufacturer to establish the
identity and purity of the culture from which the unformulated
MPCA is produced, the method of manufacture, and techniques used
to ensure a uniform or standardized product. The integrity of
the product as determined by specific and sensitive chemical,
serological or biological tests is to be demonstrated. If the
test is not a recognized standard test, a detailed description of
the test together with information regarding precision, specificity,
interfering substances, accuracy, and sensitivity must be provided.
A sample of registered MPCAs is to be maintained on deposit in
a nationally recognized culture collection. A large initial batch
could be preserved in small aliquots to serve as seed stock for
subsequent batches. This would minimize contamination and variation
(chromosomal and extrachromosomal) caused by serial passage. If
the MPCA produces a toxin, this may need to be monitored as a form
of quality control.
151A-12 Discussion of Formation of Unintentional Ingredients
As required by 40 CER 158.740, each registration
application shall include the following information:
(a) Theoretical discussion. The theoretical discussion
concerns the formation of each substance, aside from the control
agents and intentionally added, chemically characterized active
and inert ingredients, that might reasonably be present in the
pesticide product, as outlined in 61-3 of Subdivision D. Examples
of such extraneous materials are: allergens, microbial toxins,
and other metabolic products; mutant strains; microbial contaminants
with particular reference to potentially infective or antagonistic
forms; side products from chemical reactions employed in the
manufacturing process, fermentation residues from the growth of
bacteria or fungi; extraneous host residues from viruses produced
in cell cultures, whole animals, or other living forms; residues
of contaminants that remain following the purification or extraction
process; and impurities in chemicals used in the manufacturing
process. The discussion shall include the procedures used to
ensure the purity of the unformulated MPCA preparation; if purity
(within reasonable limits) cannot be achieved, then the means of
controlling contaminant levels to an acceptable limit must be
delineated.
(b) Toxic or sensitizing substances. If substances toxic
or sensitizing to humans or other nontarget mammalian species are
known or suspected to be present at any stage of the manufacturing
process, then data must be submitted to show that the substances
do not exist in the final product or exist only in quantities too
small to pose any hazard.
(c) Human or animal pathogens. Human or other nontarget
animal pathogens such as (but not limited to) Shiaella. Salmonella.
and Vibrio must not be present at hazardous levels in the techni-
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Subdivision M
cal grade of the active ingredient. If the production method can
support the growth of human or animal pathogens, each production
batch must be tested for their presence. Each application for regis-
tration of a manufacturing-use product or end-use product should con-
tain an analysis of all human or animal pathogens that might be present
at potentially hazardous levels in the product before formulation.
The application should propose methodology for detecting and/or
elimiiiating these from the product. For example, the method prescribed
in 40 CFR 180.1011 to monitor each production batch of Bacillus
thurincriensis for the pathogen Bacillus anthracis involves sub-
cutaneous injection of at least 1 million viable microorganisms
or spores into each of 5 laboratory mice weighing 17 to 23 grams.
Such tests shall show no evidence of infection or injury in the
test animals when observed for 7 days following injection.
Methods should be proposed for controlling any hazardous conta-
mination detected in the product. Discarding of a contaminated
production batch or treating the formulation may be equally acceptable
methods for controlling the contaminating microorganisms.
151A-13 Analysis of Samples
A report on the results of preliminary analysis are
required by 40 CFR 158.740 to support the registration of each
manufacturing-use product and those end-use products produced by
an integrated formulation system.
The guidelines of 62-1 of Subdivision D regarding
the analysis of samples shall apply, with the exception that a
quantitative serological or other appropriate test for the MPCA
may be substituted for the chemical analysis or may be used for
confirmation.
The Agency shall entertain waiver requests for analysis
down to 0.1% (w:w) based upon the nature of the organism, the manu-
facturing process, and known or potential ability of the MPCA to
produce a toxin or other residue of concern to the Agency.
151A-14 [Reserved]
151A-15 Certification of Ingredient T.iTm'-K«a
As required by 40 CFR 158.740, each registration must
be supported by a certification of ingredient limits. The guide-
lines of 62-2 and 62-3 of Subdivision D regarding certification
of limits and analytical methods to verify certified limits,
respectively, shall apply. The limits for microbial agents (MPCAs
and contaminants) should be expressed as: (i) MPCA units per unit
weight or volume; (ii) international units of potency per unit
weight; and (iii) weight percent of product. Items (i) and
(ii) may be determined using biological, genetic, biochemical,
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Pesticide Assessment Guidelines
serological or other appropriate tests.
Note that two or more methods to verify the certified
limits of the MPCA(s) and any micrcbial impurities may be required.
Plate counts (colony- or plague-forming units/unit weight or
volume) or infectivity assays may be necessary for quantitation
and to allow preliminary identification. Antibiotic resistance
bioassays may eliminate some contamination as well as provide
additional identification support. In some cases, especially in
the case of genetically altered MPCA's, absolute identification
can be achieved only through the use of one or more of the various
immunological methods (such as enzyme-linked imrnunosorbent assay)
or molecular probe methods (such as the Southern hybridization
procedure or restriction endonuclease mapping) . Methods must be
specific enough to determine the MPCA in the presence of revertants/
mutants and contaminants that may have formed or been introduced
during the replication/manufacturing process.
151A-16 Phsical and Chemical Properties
(a) Wh^n reqmnpd. Data on physical and chemical properties
are required to support the registration of each TGAI, manufacturing-
use product, and end-use product. See 40 CFR 158.50 and 158.740
to determine whether these data must be submitted.
(b) Substances tested. Table 1 presents the relevant data
pertaining to physical and chemical properties for MPCA preparations.
Sections 63-1 through -21 of Subdivision D should be consulted
regarding the conduct and the specific provisions of the tests.
151A-17 Submittal of Samples
When required by the Agency, as provided in 40 CER
158.740, the applicant shall submit a sample of the technical
grade of the active ingredient, manufacturing-use product, or end-
use product. Directions for storage are to be provided including
details involving safety and sample integrity. When required by
the Agency, the applicant shall submit a sample of any additional
substances in the product as listed in 151A-10(c) of this subdivision.
The samples should be sent to: U. S. Environmental Protection
Agency, Pesticides and Industrial Chemicals Repository, 2 Triangle
Drive, Research Triangle Park, NC 27709, or other facility, as
designated.
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Subdivision M
Table 1. SUMMARY OF PHYSICAL AND CHEMICAL DATA FOR MICROBIAL PEST
CONTROL AGENTS
Color
Physical state
Odor
Density or specific
gravity
pH
Stability3
Storage Stability
Viscosity
Miscibility
Technical grade
of the active
inoredient
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Corrosion characteristics No
Test Substance
Manufacturing-
use product
Yes
Yes
Yes
Yes
Yes
No
Yes
End-use
product
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes Yes
(liquids only)
Yes Yes
(emulsifiable liquids only)
Yes
(when packaged
plastic or paper
Yes
in metal,
containers)
aRefers to the stability of the MPCA to sunlight, water (such as pH
5, 7, and 9), air, normal and elevated temperatures, and metals and
their ions.
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Pesticide Assessment Guidelines
Series 152A: TOXICOLOGY GUIDELINES FOR MICRQBIAL PEST
CONTROL AGENTS
152A-1 Overview
(a) Scope of toxicology requiTgments. This section series
sets forth guidelines for testing to determine the potential for
detrimental effects to humans and domestic animals caused by microbial
pest control agents.
The testing of MPCAs for possible effects on humans and domestic
animals is performed in a sequence of three tiers. The general tier
sequence and studies involved for MPCAs are outlined in the Appendix to
the document.
(b) Purpose. The purpose of this section is to consider the
toxicological concerns that the Agency has for MPCA preparations, and
to discuss these concerns in relation to the tiered testing scheme for
evaluating pathogenic and toxic effects. Major concerns of the Agency
for MPCA preparations that relate to Toxicology are the following:
(1) pathogenicity of the MPCA and of microbial contaminants;
(2) infectivity/unusual persistence of the MPCA and of microbial
contaminants;
(3) toxicity of the MPCA, of microbial contaminants, and of
preparation by-products.
(c) Tier I testing. The overall purpose of the Tier I studies is
to provide a toxicological evaluation of a MPCA preparation with respect
to the above three endpoints. The Agency recognizes that pathogenicity,
infectivity, and toxicity comprise a complex set of microorganism: host
interactions which may not be easily resolved as independent endpoints
in all studies. However, it is believed that, in most cases, the data
from the Tier I battery of tests will provide a fairly clear evaluation
of the potential risks of the MPCA due to pathogenicity, infectivity,
and toxicity.
The following Tier I studies are considered appropriate for testing of
MPCA preparations:
(1) Acute oral toxicity/pathogenicity study;
(2) Acute pulmonary toxicity/pathogenicity study;
(3) Acute intravenous toxicity/pathogenicity study.
The following Tier I studies are considered appropriate primarily
to evaluate the toxicity of chemical components of an MPCA prepa-
ration (but are performed on the complete MPCA preparation):
(4) Acute dermal toxicity study;
(5) Primary eye irritation study;
(6) Reporting of hypersensitivity incidents.
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Subdivision M
The final Tier I study is specific to viruses and is designed to
evaluate adverse effects of any viral pesticide control agent to
mammalian cell lines:
(7) Cell culture tests with viral pest control agents.
Detailed interim protocols for viral testing nave been developed
by EPA's Office of Research and Development. Although these proto-
cols have not been completely validated, they are available on
request from EPA in order to provide additional guidance for appli-
cants and testing laboratories.
(d) Tier II testing. Tier H testing is designed to examine
the situation where Tier I testing indicates that the MPCA exhibits
infectivity or toxicity without any evidence of pathogenicity.
(1) Infectivity/unusual persistence. Data from acute toxicity/
pathogenicity studies may indicate that the MPCA is able to infect test
animals without eliciting definite signs of pathogenicity or toxicity,
or the MPCA may be observed to persist in test animals for periods
longer than would normally be expected. The Agency considers that
these observations are sufficient to warrant subchronic testing, Tier
II, in order to determine whether repeated exposure to the MPCA preparation
is sufficient to cause toxic or pathogenic effects. Subchronic studies
also may be required if an MPCA preparation is unable to be sufficiently
freed of contaminating microorganisms, or contaminating microorganisms
are insufficiently characterized, or there is sufficient reason to
believe that ndcrobially-produced toxins exist in the preparation, and
these toxins would not exert effects in test animals after acute ex-
posure in the Tier I studies.
The Agency does not choose to set statistical limits to delineate
the meaning of "significant infectivity" or to define "unusual persistence"
by selecting minimum periods of time for clearance of a microorganism
from a test animal. It is believed that these terms are best defined
in the context of data that are obtained from the battery of Tier I
studies, and in consideration of the type of microorganism and the
route of exposure. In general, the MPCA can be considered as "infective"
if evidence is obtained that shows the microorganism can cross or evade
natural host barriers to infection. Evidence of replication of the
microorganism in the host also bears on the interpretation of whether
it is infective for the test animal.
The Agency recognizes that certain forms of microorganisms (e.g.
spores) may be cleared from the host animal at a rate slower than vege-
tative forms of the same microorganism. In addition, in the Tier I tests,
it is expected that certain MPCAs may be isolated from the test animal,
even at the end of the recommended observation periods. Such isolations
should not automatically trigger Tier II testing, the data will be inter-
preted in light of the decline curves generated, and also in light of
any evidence that the MPCA replicates in the host animal.
(2) Toxicitv. Toxicity of a MPCA preparation to test animals
may be caused by substances produced by, or which are constituents of,
the MPCA or any contaminating microorganism; or by substances that are
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Pesticide Assessment Guidelines
present as non-biological components of the preparation. The Tier I
toxicity/infectivity studies are designed to detect acute toxic effects
of biological or non-biological cxsnponents of a MPCA preparation,
in the absence of signs of pathogenicity or infectivity. The acute
dermal toxicity study is designed primarily to evaluate toxic effects
due to chemical components of the MPCA or to non-biological components
of a MPCA preparation. The Agency believes that only rarely would it
be expected that a microorganism could bypass the skin barrier, or
infect epithelial cells, of a healthy host animal solely by virtue of
being placed in contact with the skin. An acute intravenous toxicity/
pathogenicity study has been placed in Tier I, to evaluate possible
adverse effects of a MPCA preparation once the skin barrier is
deliberately bypassed.
If toxic effects are observed in the Tier I studies, in the absence
of signs of infectivity or pathogenicity, then a Tier II acute toxicity
study normally is required, with the toxic components of the MPCA
preparation being used as the dosing material. Additional studies
designed solely for evaluating toxicity effects of MPCA preparations
are not included in the Tier II or Tier III testing scheme for microbial
pesticides because appropriate test protocols already exist in Subdiv-
ision F guidelines for testing of conventional chemical pesticides, or
in the portion of these guidelines for testing of biochemical pest
control agents. These guidelines can be consulted, if required, to
provide a full evaluation of toxicity due to components of a MPCA
preparation.
(e) Tier III testing. In general, if it is determined from Tier
I test results that the MPCA is pathogenic for the test animals, then
the Agency is to be consulted to determine the next appropriate course
of action. It has been the Agency's experience, to date, that recognized
mammalian pathogens have not been considered as likely candidates for
registration as pesticides. However, it is not inconceivable that
recognized pathogens of mammals may be considered for development as
MPCAs, for instance, as rodenticides. In such cases, careful consider-
ation and thorough evaluation for pathogenic effects of the MPCA for
nontarget mammals is to be provided. Also, the Agency realizes that
certain recognized mammalian pathogens that have been rendered non-path-
ogenic by recombinant ENA or equivalent techniques, may be considered
for use as microbial pesticides. In these cases, the effectiveness of
the disarming procedure will have to be clearly established.
Viruses and protozoa require special attention for the following
reasons:
(i) viruses are obligate intracellular parasites, and protozoa may
be obligate or facultative intracellular parasites;
(ii) these intracellular parasites may be less amenable to sufficient
taxonomic identification;
(iii) preparations of these MPCAs that are free of contaminating
organisms or cellular debris may be difficult to obtain; and,
(iv) they may be present as contaminants in preparations of other MPCAs.
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Subdivision M
The studies that comprise Tier III primarily are designed for
evaluating the potential of MPCAs, or microbial contaminants of MPCA
preparations, that are recognized to be intracellular parasites of
mammalian cells, or are shown to be, by data resulting from conducting
the lower tier studies. It is expected that registrants would not
ordinarily pursue registration of such MPCAs. The Tier III studies also
may be appropriate for evaluating known mammalian parasites that have
been disarmed of pathogenic traits by use of recombinant ENA or
equivalent techniques.
The Agency is concerned about proper characterization and identi-
fication of microorganisms that are present as contaminants in MPCA
preparations, including formulations and newly-produced batches of any
MPCA. Appropriate quality control procedures and appropriate biological
analyses shall be employed so to ensure that adverse human and
mammalian health effects will not occur from use of MPCA products
contaminated with other microorganisms. Also, records of biological
analyses shall be maintained and made available to the Agency if
required.
(4) Other issues.
(i) Identification and characterization of MPCAs. A sufficient
and appropriate taxonomic evaluation of the MPCA is required for an
initial assessment of potential adverse health effects to mammals,
including humans. In addition to the requirements of 151-20 to -27,
data on the effects of temperature range on growth of the MPCA also
are useful in determining whether the MPCA may survive or grow at
mammalian body temperatures.
If the MPCA is genetically altered, then the methods used
to derive the product are to be described in detail; and the correct
structure of the gene construct in the engineered MPCA product is
to be confirmed. The requirements set forth in 151A-10 to-17 for
genetically altered MPCAs also must be met.
(ii) Allergy/hypersensitivity. The Agency believes that to
require reporting of observed allergic responses of humans to MPCA
preparations is sufficient to adequately address health problems
due to types of these reactions. Based on the information provided
in such reports, the Agency would recommend adequate precautions in
handling the material. In some instances, the MPCA will be a
recognized common allergen. In these cases, the Agency may require
proper precautionary label statements. The Agency strongly recommends
that appropriate respiratory tract coverings be worn when there
might be accidental exposure to aerosols of MPCA preparations. The
purpose of the recommended coverings is to minimize unnecessary
stimulation of the respiratory system.
(iii) Eye irritation/injury. Appropriate eye protection is
recommended when exposure (including accidental exposure) to aerosols
of the MPCA preparation may occur. If it is demonstrated (e.g., by
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Pesticide Assessment Guidelines
appropriate label precautionary statements) that workers/applicators' s
eyes will be protected by appropriate coverings (e.g., goggles) then the
primary eye irritation study (152A-14) may not be required.
152A-2 through -9 [Reserved]
Group 1: Tier I Testing.
152A-10 Acute oral toxicity/pathocfenicity study with microbial
pest control agents [(MPCA)s]; Tier I.
(a) When required. Acute oral toxicity/pathogenicity data are
required by 40 CER 158.740 to support the registration of each
manufacturing-use product and each end-use product, and the technical
grade of each active ingredient.
(b) Purpose. In the assessment and evaluation of the toxic or
pathogenic characteristics of a MPCA, determination of acute oral
toxicity/pathogenicity usually is an initial step. It provides
information on health hazards likely to arise from a single exposure
by the oral route. The purpose of the acute oral study is to provide
initial information on the toxicity, infectivity, and pathogenicity
of a MPCA using a single high dose exposure and an adequate post-
exposure observation period.
(c) Definitions.
(1) "Acute oral toxicity11 and "acute oral pathogenicity" are
the adverse effects occurring from the oral administration of a
single dose of a MPCA. Acute oral toxicity also may be the adverse
effects occurring from the oral administration of a microbially
produced substance, or from other ingredients in any test substance.
(2) "Dose level" is the amount of MPCA administered. It is
expressed as units of the microorganism administered per test animal.
(3) "Units of MPCAs". One unit of representative MPCA groups
usually would be defined as follows:
(i) vegetative bacterium: a single, viable organism, and
usually the entity that produces a single colony forming unit (CFU)
on an appropriate semisolid growth medium.
(ii) bacterial or fungal spore, bacterial or protozoan cyst:
an intact individual spore or cyst as determined microscopically,
and usually the entity that produces a single CFU on appropriate
germination medium.
(iii) fungal mycelium: 10"~9 gram dry weight or, after stan-
dardized preparatory procedures, a mycelium-producing entity on
semi-solid growth medium.
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Subdivision M
(iv) virus: an intact, complete virion or a polyhedral body
as determined by electron microscopy, and, usually the entity that
produces an infective unit (IU) on appropriate host cells or tissues.
(v) protozoa: an intact vegetative organism, spore, or cyst
of the members in the various classes of this phylum.
Due to the wide diversity of forms among microorganisms, other
definitions of a unit of a MPCA may be equally appropriate.
(d) Principles of the test method. The test MPCA is adminis-
tered orally by gavage in a single high dose to experimental animals.
Subsequent observations of effects and deaths are made and rate of
clearance of the MPCA is estimated. Animals that die during the
test are necropsied, and at the conclusion of the test the surviving
animals are sacrificed and necropsied. Infectivity of the MPCA is
evaluated periodically during the test, and at the conclusion of the
test.
(e) Substance to be tested.
(1) The technical grade of each active ingredient shall be
tested to support the registration of each manufacturing-use product,
and each end-use product.
(2) The form (e.g. vegetative cell, spore, cyst, virion) of
the MPCA used in testing should be equivalent to the form that is
intended for registration or application. To the extent possible,
the test MPCA also should be equivalent to the MPCA intended for
registration or application with respect to stage of growth, possession
of organelles and appendages and expression of phenotypic traits
(including products from intentionally introduced genes). If signi-
ficant exposure to other forms of the MPCA are expected, or if
changes in form of the MPCA occur in the host, then these forms
also may have to be tested.
(f) Characteristics of the test MPCA. The test MPCA should
be thoroughly characterized as required in 151A of these guidelines.
(g) Test procedures.
(1) Animal selection.
(i) Species and strain. Although several mammalian test
species may be used, the mouse or the rat are the preferred rodent
species. Commonly used laboratory strains should be employed. If
another species is used, justification/reasoning for the alternative
selection should be provided. All test animals should be free of
parasites or pathogens. Females should be nulliparous and
non-pregnant.
(ii) Age. Young adult animals should be used. The weight
variation of animals used in a test should not exceed + 20 percent
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Pesticide Assessment Guidelines
of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex are required.
(iv) Numbers. At least 6 animals (three animals of each sex)
should be used. A sufficient number of additional animals should
be used to allow for interim sacrifice for infectivity determinations,
and also, for control groups.
(2) Control groups.
(i) A concurrent untreated control group of four animals/sex
is required. Half of the animals in the control group (i.e., two
animals/sex) should be housed separately from the test group of
animals dosed with MPCA, and the remainder of the control animals
(the "shelf control" group) should be housed with the dosed animals.
(ii) A separate vehicle control group is not required except
when the toxicity of the vehicle is unknown.
(iii) Control groups dosed with inactivated MPCA (i.e., rendered
incapable of reproduction or germination or excystment) may prove use-
ful to evaluate toxic properties of the MPCA. Inactivation should
be done by a means that allows for reasonable maintenance of the
structural integrity of the MPCA.
(3) Dosing.
(i) Dose level. One dose level of at least 108 units of the
MPCA per test animal should be used. If a dose level of at least
108 units per test animal is not used, a justification/explanation
must be provided.
(ii) Vehicle. The recommended vehicle for the technical grade
of each active ingredient is one that allows for maintenance of
viability, or germination capability, or excystment capability/ or,
for intracellular parasites, infection capability in a suitable
host.
(iii) Volume. The maximum volume of liquid that can be adminis-
tered at one time depends on the size of the test animal. In rodents,
the volume should not exceed 2 ml/100 g body weight. Variability in
test volume should be minimized.
(iv) Dose quantification. Techniques used to quantify the
units of MPCA in any dose will depend on the group of microorganisms
to which the MPCA belongs. Where possible, determinations of viable,
or potentially viable, or infective units in each dose should be
made. A measurement of metabolism associated with a defined biomass
may be the preferred technique for quantification of mycelial forms
of MPCAs. Quantification should be done concurrently with testing.
(4) Exposure.
(i) The test substance should be administered in a single dose
by gavage, using a stomach tube or suitable intubation cannula.
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Subdivision M
(ii) Animals should be fasted overnight prior to test subs-
tance administration. After the substance has been administered,
food may be withheld for a further 3-4 hours.
(iii) If a single dose is not possible, the dose may be given
in smaller portions over a period not exceeding 24 hours. Where a
dose is administered in fractions over a period, it may be necessary
to provide the animals with food and water, depending on the length
of the period.
(5) Observation period. Ihe observation period should be at
least for 21 days after dosing. However, the duration of observation
should not be fixed rigidly. It should be determined by the type
of MPCA administered and its rate of clearance from the test animals.
Duration of the observation period also would depend on the time at
which signs of toxicity and pathogenicity appear and disappear, and
the time to death of the animals.
(6) Observation of animals.
(i) A careful clinical examination of all animals should be made at
least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy
of, and MPCA enumeration from those animals found dead, and isolation of weak or
moribund animals.
(iii) Cageside observations should include, but not be limited to,
changes in:
(A) the skin and fur;
(B) eyes and mucous membranes;
(C) respiratory system;
(D) circulatory system;
(E) autonomic and central nervous system;
(F) somatomotor activity; and,
(G) behavior pattern.
(H) Particular attention should be directed to observation of
tremors, convulsions, diarrhea, lethargy, salivation, sleep and coma.
(iv) Individual weights of animals should be determined shortly
before the test material is administered, weekly thereafter, and at death
or at interim or final sacrifice. Changes in weight should be calculated
and recorded when survival exceeds one day.
(v) The time of death should be recorded as precisely as possible.
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Pesticide Assessment Guidelines
(7) Gross pathology. A gross necropsy of all animals should be
performed at the time of death or at interim or final sacrifice. All
gross pathological changes should be recorded.
(8) Clearance of MPCA. Feces from test animals should be collected
soon after dosing and frequently during the study and examined for the
presence of the MPCA to estimate clearance of the MPCA after oral admi-
nistration. Methods (e.g. immunological assays, ENA probes) other than
those used for quantification of MPCAs in each dose may prove useful.
Recovery values and detection and sensitivity limits should be determined
and reported for each technique used.
or persistence should be assessed by using sensitive techniques to determine
the presence of the MPCA in tissues, organs, and body fluids. Methods
other than those used for quantification of MPCAs in each dose may prove
useful. Recovery values and detection and sensitivity limits should be
determined and reported for each technique used. Methods selected for MPCA
enumeration should, if possible, allow for detection of microbial replication
(10) Interim sacrifice. For evaluating infectivity and clearance, the
MPCA should be enumerated from tissues, organs, and body fluids of three
treated animals per sex, sacrificed at three days after, and then at one
week intervals after dosing. The number of interim sacrifice periods re-
quired will depend on the nature of the test microorganism, and should be
sufficient to adequately establish a pattern of clearance. The MPGA also
should be enumerated from the tissues, organs, and body fluids of the
"shelf control" group at final sacrifice.
(11) Tissues, organs, and body fluids, (i) For infectivity and
persistence determinations, the MPCA should be enumerated from the kidney,
brain, liver, lung, spleen, blood, representative lymph nodes, and, where
appropriate, from lesions and from the injection site, (ii) Other tissues,
organs, and body fluids may have to be examined as indicated by the nature
of any toxic and pathogenic effects observed.
(i) Data and reporting.
(1) Treatment of results. In addition to the information recom-
mended by 150A-4 of this subdivision and 80-4 of subdivision F, the
test report should include the following information:
(i) number of animals at the start of the test;
(ii) time of death of individual animals;
(iii) number of animals displaying other signs of toxicity and
pathogenicity;
(iv) description of toxic and pathogenic effects;
(v) definition for one unit of the MPCA used, and the units/test
animal in the dosing material;
(vi) body weights and time taken;
(vii) necropsy findings;
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38
Subdivision M
(viii) pathology findings/ when performed;
(ix) infectivity/persistenoe findings;
(x) estimate of rate of MPCA clearance;
(xi) description of all enumeration methods used for MPCA. detection
and quantification; and,
(xii) verification that each enumeration method is sufficiently sen-
sitive to serve as a useful quantitative assay for the MPCA in tissues,
organs, and body fluids.
(2) Evaluation of results. An evaluation should include the
relationship, if any, between exposure to the test substance and the
incidence and severity of all abnormalities, including;
(i) behavioral abnormalities;
(ii) clinical abnormalities;
(iii) gross lesions;
(iv) body weight changes;
(v) mortality;
(vi) toxicity;
(vii) infectivity; and,
(viii) pathogenicity.
»
(j) Tier progression
(1) If persistent or significant signs of pathogenicity of the
MPCA. for the test animals is observed in Tier I, acute oral toxicity/
pathogenicity tests may be required in non-rodent animal species.
Consultation with the Agency to discuss further testing requirements
is recommended.
(2) If toxin production by the MPCA is suspected, or if toxin
production is indicated by significant or persistent signs of toxicity
in the test animals, in the absence of signs of infectivity or
pathogenicity, then;
(i) the toxic component(s) of the dosing material is (are) to
be identified, and to a practical extent, isolated.
(ii) an acute toxicity study (152A-20) is to be conducted with
the toxic component (s).
(3) If significant infectivity or unusual persistence of the
MPCA. is observed in the absence of signs of toxicity or pathogenicity,
then a subchronic (90-day) study ( 152A-21) is required.
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Pesticide Assessment Guidelines
152A-11 Acute dermal toxicity study with microbial pest control
agents r(MPCA)s1; Tier I.
(a) When required. Acute dermal toxicity data are required by
40 CER 158.740 to support the registration of each manufacturing-use
product and each end-use product, and the technical grade of each
active ingredient.
(b) Purpose. Acute dermal toxicity data provide information
on health hazards likely to arise from a single dermal application
of soluble or particulate chemicals associated with a preparation
of the MPCA, and/or associated with other ingredients in formulations
of the MPCA, and/or associated with products from genetic material
intentionally introduced into the MPCA.
(c) Definitions.
(1) "Acute dermal toxicity" is the adverse effect occurring
during or following a 24-hour dermal exposure to a single dose of
a test substance.
(2) "Dose level" is based on the amount of MPCA administered.
It is expressed as;
(i) units of the microorganism applied at each test site on
the test animal, and as;
(ii) dry weight of applied test substance at a single test site
per kilogram body weight of test animal.
(3) "Units of MPCAs". One unit of representative MPCA groups
usually would be defined as follows:
(i) vegetative bacterium: a single, viable organism, and
usually the entity that produces a single colony forming unit
(CFU) on an appropriate semisolid growth medium.
(ii) bacterial or fungal spore, bacterial or protozoan cyst:
an intact individual spore or cyst as determined microscopically,
and usually the entity that produces a single CPU on appropriate
germination medium.
(iii) fungal mycelium: 10"9 gram dry weight or, after
standardized preparatory procedures, a mycelium-producing entity on
semi-solid growth medium.
(iv) virus: an intact, complete virion or a polyhedral body
as determined by electron microscopy, and, usually the entity that
produces an infective unit (IU) on appropriate host cells or tissues.
(v) protozoa: an intact vegetative organism, spore, or cyst
of the members in the various classes of this phylum.
Due to the wide diversity of forms among microorganisms, other
definitions of a unit of a MPCA may be equally appropriate.
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Subdivision M
(d) Principles of the test method. The MPCA in each for-
mulation to be tested is applied in a single high dose to the skin
of experimental animals. Subsequently, observations of effects and
deaths are made. Animals that die during the test are necropsied,
and at the conclusion of the test, the surviving animals are sacrificed
and necropsied as indicated by the nature of the toxic effects
observed.
(e) Substance to be tested.
(1) The manufacturing-use product shall be tested, to support
the registration of each manufacturing-use product.
(2) The end-use product shall be tested to support the regis-
tration of each end-use product.
(3) The form (e.g. vegetative cell, spore, cyst, virion) of
the MPCA used for preparation of the dosing material should be
equivalent to the form that is intended for registration or applica-
tion. To the extent possible, the test MPCA also should be equivalent
to the MPCA intended for registration or application with respect
to stage of growth, possession of organelles and appendages, and
expression of phenotypic traits (including products from intentionally
introduced genes). If significant exposure to other forms of
the MPCA are expected, then these forms also may have to be tested.
(f) Characteristics of the test MPCA. The test MPCA should
be thoroughly characterized as required in section 151A of these
guidelines.
(g) Test procedures.
(1) Animal selection.
(i) Species and strain. Although several mammalian test
species may be used, the albino rabbit is the preferred species.
Commonly used laboratory strains should be employed. If another
species is used, the investigator should provide justification/rea-
soning for the alternative selection. All test animals should be
free of parasites or pathogens. Females should be nulliparous and
non—pregnant.
(ii) Acre. Young adult animals should be used . The weight
variation of animals used in a test should not exceed ± 20 percent
of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex with healthy
intact skin are recommended.
(iv) Numbers. At least 10 animals (five animals of each sex)
should be used.
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Pesticide Assessment Guidelines
(2) Control groups, (i) Neither a concurrent untreated nor
vehicle control group are required except when the toxicity of
the vehicle is unknown.
(3) Dosing, (i) Dose level. The test substance should be
applied at 2 grams/test animal. If a dose level of less than 2 grams/
test animal is used, then a justification/explaination must be provided.
(ii) Vehicle. Where necessary, the formulation to be tested
is suspended in a suitable vehicle. An aqueous solution should be
used. The recommended vehicle for the end-use product usually is
the same material in which the MPCA will be mixed, suspended, or
diluted for application.
(iii) Volume. Ihe moisture content of the test material should
not be excessive, but should be sufficient to prevent significant
drying of the test, material during the exposure period, and to
insure good contact with the skin.
(iv) MPCA quantification. The numbers of MPCAs in the dose mat-
erial should be enumerated. Techniques used to quantify the units
of MPCA in any dose will depend on the group of microorganisms to
which the MPCA belongs. Where possible, determinations of viable,
or potentially viable, or infective units in each dose should be
made. A measurement of metabolism associated with a defined biomass
may be the preferred technique for quantification of mycelial forms
of MPCAs. Quantification should be done concurrently with testing.
(4) Exposure duration. The exposure duration should be for
approximately 24 hours.
(5) Observation period. The observation period should be at
least for 14 days, but should not be fixed rigidly. It should be
determined by the toxic reactions, rate of onset and length of
recovery period, and thus may be extended when considered necessary.
The time at which toxicity signs appear and disappear, and their
duration are important.
(6) Preparation of animal skin.
(i) Approximately 24 hours before the test, fur should be
removed from the dorsal and ventral area of the trunk of each test
animal by clipping or shaving.
(ii) Not less than 10 percent of the body surface area should
be cleared for application of the test substance. The weight of
the animal should be taken into account when deciding on the area
to be cleared and on the dimensions of the covering.
(7) Application of the test substance.
(i) The test substance should be applied uniformly over an
area which is approximately 10 percent of the total body surface
area.
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Subdivision M
(ii) The test substance should be held in contact with the
skin with porous gauze and a non-irritating tape throughout a 24-hour
exposure period. Ihe test site further should be covered in a
suitable manner to retain the gauze dressing and test substance and
ensure that the animals cannot ingest the test substance. Restrainers
may be used to prevent the ingestion of the test substance, but
complete immobilization is not recommended.
(iii) At the end of the exposure period, residual test substance
should be removed, where practical, using water.
(8) Observation of animals.
(i) A careful clinical examination should be made at least
once each day.
(ii) Cageside observations should include, but not be limited
to, changes in:
(A) the skin (including signs of irritation) and fur;
(B) eyes and mucous membranes;
(C) respiratory system;
(D) circulatory system;
(E) autonomic and central nervous system;
(F) somatomotor activity; and,
(G) behavior pattern.
(H) Particular attention should be directed to observation of
tremors, convulsions, diarrhea, lethargy, salivation, sleep and
coma.
(iv) Individual weights of animals should be determined
shortly before the test material is administered, weekly thereafter,
and at death or at final sacrifice. Changes in weight should be
calculated and recorded vfaen survival exceeds one day.
(v) The time of death should be recorded as precisely as
possible.
(vi) At the end of the 24 exposure period, and daily thereafter,
any signs of skin irritation should be recorded and scored.
(9) Gross pathology. Consideration should be given .to per-
forming a gross necropsy of all animals if indicated by the ap-
pearance of toxic effects. If done, all gross pathological changes
should be recorded.
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Pesticide Assessment Guidelines
(i) Data and reporting.
(1) Treatment of results. In addition to the information
recommended by 150A-4, the test report should include the follow-
ing information:
(i) number of animals at the start of the test;
(ii) time of death of individual animals;
(iii) number of animals displaying other signs of toxicity;
(iv) description of toxic effects;
(v) definition for one unit of the MPCA used, and the units
in the dosing material;
(vi) dry weight and net weight determinations of the test
material applied per kilogram body weight of the test animal;
(vii) body weights and time taken;
(viii) necropsy findings, when performed; and,
(ix) pathology findings, when performed;
(2) Evaluation of results. An evaluation should include the relationsh
if any, between the animals exposed to the test substance and the incidence a
severity of all abnormalities, including;
(i) behavioral abnormalities;
(ii) clinical abnormalities;
(iii) skin lesions and skin irritation;
(iv) body weight changes;
(v) mortality; and,
(vi) toxicity.
(j) Tier progression.
(1) If evidence of significant and/or persistent toxicity are
observed, then the toxic component (s) of the dosing material are to
be identified, and to a practical extent, isolated.
(i) An acute toxicity study (152A-20) is to be conducted with
the toxic components.
(2) If no toxic effects are observed, then no further testing
is required.
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Subdivision M
152A-12 Acaite pulroonarv toxjcitv/pathocrenicitv study with
microbial pest control agents ffMPCA)s1; Tier I.
(a) When required. Acute pulmonary toxicity/pathogenicity
data are required by 40 CER 158.740 to support the registration
of each manufacturing-use product and each end-use product/ and the
technical grade of each active ingredient.
(b) Purpose. Acute pulmonary toxicity/pathogenicity data
provide information on health hazards likely to arise from a single
exposure by the pulmonary route. Hie purpose of the acute pulmonary
study is to provide initial information on the toxicity, infectivity,
and pathogenicity of a MPCA using a single high dose exposure and
an adequate post-exposure observation period.
(c) Definitions.
(1) "Acute pulmonary toxicity" and "acute pulmonary pathogenicity"
are the adverse effects occurring from intranasal or intratracheal
administration of a dose of a MPCA . Acute pulmonary toxicity also
may be the adverse effects occurring from intranasal or intratracheal
a microbially produced substance.
(2) "Dose level" is the amount of MPCA administered. It is
expressed as units of the microorganism administered per test animal.
(3) "Units of MPCAs". One unit of representative MPCA groups
usually would be defined as follows:
(i) vegetative bacterium: a single, viable organism, and
usually the entity that produces a single colony forming unit (CFU)
on an appropriate semisolid growth medium.
(ii) bacterial or fungal spore, bacterial or protozoan cyst:
an intact individual spore or cyst as determined microscopically,
and usually the entity that produces a single CFU on appropriate
germination medium.
(iii) fungal mycelium: 10~9 gram dry weight or, after standardized
preparatory procedures, a mycelium-producing entity on semi-solid
growth medium.
(iv) virus: an intact, complete virion or a polyhedral body
as determined by electron microscopy, and, usually the entity that
produces an infective unit (IU) on appropriate host cells or tissues.
(v) protozoa: an intact vegetative organism, spore, or cyst
of the members in the various classes of this phylum.
Due to the wide diversity of forms among microorganisms, other
definitions of -a unit of a MPCA may be more appropriate.
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Pesticide Assessment Guidelines
(d) Principles of the test method. The test MPCA is administered
by intranasal or intratracheal routes in a single high dose to
experimental animals. Subsequent observations of effects and deaths
are made, and the rate of clearance of the MPCA is estimated. Animals
that die during the test are necropsied, and at the conclusion of
the test the surviving animals are sacrificed and necropsied. Infec-
tivity of the MPCA is evaluated periodically during the test and at
the conclusion of the test.
(e) Substance to be tested.
(1) The technical grade of each active ingredient shall be
tested to support the registration of each manufacturing-use product,
and each end-use product.
(2) In some formulations, the aerodynamic mean diameter of the
particles may be so large as to render pulmonary testing impractical, or
unwarranted. In such cases, the test substance may consist only of the
purest infective form of the microorganism. However, justification/
reasoning must be provided by the investigator to support a request
for exempting specific technical grade preparations from testing by
intranasal or intratracheal dosing. In other cases, the aerodynamic
mean diameter of the particles may be sufficiently large as to
warrant intratracheal exposure versus intranasal instillation.
(3) The form (e.g. vegetative cell, spore, cyst, virion) of
the MPCA used in testing should be equivalent to the form that is
intended for registration or application. To the extent possible,
the test MPCA also should be equivalent to the MPCA intended for
registration or application with respect to stage of growth, possession
of organelles and appendages and expression of phenotypic traits
(including products from intentionally introduced genes). If sig-
nificant exposure to other forms of the MPCA are expected, or if
changes in form of the MPCA occur in the host, then these forms
also may have to be tested.
(f) Characteristics of the test MPCA. The test MPCA should
be thoroughly characterized as required in section 151A of these
guidelines.
(g) Test procedures.
(1) Animal selection.
(i) Species and strain. Although several mammalian test
species may be used, the mouse or the rat are the preferred rodent
species. Commonly used laboratory strains should be employed. If
another species is used, the investigator should provide justifi-
cation/reasoning for the alternative selection. All test animals
should be free of parasites or pathogens. Females should be nul-
liparous and non-pregnant.
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Subdivision M
(ii) Age. Young adult animals should be used. The weight
variation of animals used in a test should not exceed + 20 percent
of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex are required.
(iv) Numbers. At least 10 animals (five animals of each sex)
should be used. A sufficient number of additional animals should
be used to allow for interim sacrifice for infectivity determinations,
and also for control groups.
(2) Control groups.
(i) A concurrent untreated control group is required.
(ii) A separate vehicle control group is not required except
when the toxicity of the vehicle is unknown.
(iii) Control groups dosed with inactivated MPCA (i.e., rendered
incapable of reproduction or germination or excystment) may prove
useful to evaluate toxic properties of the MPCA. Inactivation
should be done by a means that allows for reasonable maintenance of
the structural integrity of the MPCA.
(3) Dosing.
(i) Dose level. One dose level of at least 108 units of
the MPCA per test animal should be used. If a dose level of at
least 108 units per test animal is not used, a justification/ex-
planation must be provided.
(ii) Vehicle. The recommended vehicle for the technical grade
of each active ingredient is one that allows for maintenance of
viability, or germination capability, or excystment capability, or,
for intracellular parasites, infection capability in a suitable
host. The recommended vehicle for the manufacturing-use product or
end-use product is the same material in which the MPCA will be
distributed, mixed, suspended, or diluted for application.
(iii) Volume. The maximum volume of liquid that can be admi-
nistered via intranasal or intratracheal routes at one time depends
on the size of the test animal. In rodents, the volume usually
should not exceed 0.3 ml/100 g body weight. Variability in test
volume should be minimized.
(iv) Dose quantification. Techniques used to quantify the
units of MPCA in any dose will depend on the group of microorganisms
to which the MPCA belongs. Where possible, determinations of
viable, or potentially viable, or infective units in each dose
should be made. A measurement of metabolism associated with a
defined biomass may be the preferred technique for quantification
of mycelial forms of MPCAs. Quantification should be done concur-
rently with testing.
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Pesticide Assessment Guidelines
(4) Exposure.
(i) The test substance should be administered via an appropriate
delivery system in a single dose into the trachea of each test
animal, or by intranasal instillation.
(5) Observation period. The observation period should be at
least for 21 days after dosing. However, the duration of observation
should not be fixed rigidly. It should be determined by the type
of MPCA administered and its rate of clearance from the test animals.
Duration of the observation period also would depend on the time, at
which signs of toxicity and pathogenicity appear and disappear, and
the time to death of the animals.
(6) Observation of animals.
(i) A careful clinical examination should be made at least
once each day.
(ii) Additional observations should be made daily with ap-
propriate actions taken to minimize loss of animals to the study,
e.g., necropsy of, and MPCA enumeration from those animals found
dead, and isolation of weak or moribund animals.
(iii) Cageside observations should include, but not be limited
to, changes in:
(A) the skin and fur;
(B) eyes and mucous membranes;
(C) respiratory system;
(D) circulatory system;
(E) autonomic and central nervous system;
(F) somatomotor activity; and,
(G) behavior pattern.
(H) Particular attention should be directed to observation of
tremors, convulsions, diarrhea, lethargy, salivation, sleep and
coma.
(iv) Individual weights of animals should be determined
shortly before the test material is administered, weekly thereafter,
and at death or at interim or final sacrifice. Changes in weight
should be calculated and recorded when survival exceeds one day.
(v) The time of death should be recorded as precisely as
possible.
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Subdivision M
(7) Gross pathology. A gross necropsy of all animals should
be performed at the time of death or at interim or final sacrifice.
All gross pathological changes should be recorded.
(8) Clearance of MPCA. Lungs from test animals sacrificed
during the study should be examined for the presence of the MPCA to
estimate clearance of the MPCA after dosing. The first analysis should
be done on three animals/sex as soon as possible after dosing.
Methods (e.g. immunological assays, ENA probes) other than those
used for quantification of MPCAs in each dose may prove useful.
Recovery values and detection and sensitivity limits should be
determined and reported for each technique used.
(9) MPCA enumeration in tissues, organs, and body fluids.
Inf ectivity and clearance should be assessed by using sensitive techniques
to determine the presence of the MPCA in tissues, organs and body fluids.
Methods other than those used for quantification of MPCAs in each dose
may prove useful. Recovery values and detection and sensitivity limits
should be determined and reported for each technique used. Methods
selected for MPCA enumeration should, if possible, allow for detection
of microbial replication.
(10) Interim sacrifice. For evaluating infectivity and clearance, the
MPCA should be enumerated from tissues, organs, and body fluids of three
treated animals per sex, sacrificed at three days after, and then at one
week intervals after dosing. The number of interim sacrifice periods re-
quired will depend on the nature of the test microorganism, and should be
sufficient to adequately establish a pattern of clearance. The MPCA also
should be enumerated from the tissues, organs, and body fluids of the
"shelf control" group at final sacrifice.
(11) Tissues, organs and body fluids.
(i) For inf ectivity and persistence determinations, the MPCA.
should be enumerated from the kidneys, brain, liver, lungs, spleen,
blood, representative lymph nodes, caecum contents, and, where appropriate,
from lesions and the inoculation site.
(ii) Other tissues, organs, and body fluids may have to be examined
as indicated by the nature of any toxic and pathogenic effects observed.
(i) Data and reporting.
(1) Treatment of results. In addition to the information recommended
by 150A-4, the test report should include the following information:
(i) number of animals at the start of the test;
(ii) time of death of individual animals;
(iii) number of animals displaying other signs of toxicity
and pathogenicity;
(iv) description of toxic and pathogenic effects;
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Pesticide Assessment Guidelines
(v) definition for one unit of the MPCA used, and the units/test
animal in the dosing material;
(vi) body weights and time taken;
(vii) necropsy findings;
(viii) pathology findings, when performed;
(ix) infectivity findings;
(x) estimate of rate of MPCA clearance; and,
(xi) description of all enumeration methods used for MPCA
detection and quantification.
(xii) verification that each enumeration method is sufficiently
sensitive to serve as a useful quantitative assay for the MPCA
tissues, organs, and body fluids.
(2) Evaluation of results. An evaluation should include the
relationship, if any, between exposure to the test substance and
the incidence and severity of all abnormalities, including:
(i) behavioral abnormalities;
(ii) clinical abnormalities;
(iii) gross lesions;
(iv) body weight changes;
(v) mortality;
(vi) toxicity;
(vii) infectivity; and,
(viii) pathogenicity'
(j) Tier progression
(1) If persistent or significant signs of pathogenicity of the
MPCA for the test animals is observed in Tier I, acute pulmonary
toxicity/pathogenicity studies may be required in non-rodent animal
species. Consultation with the Agency is recommended to determine
the requirement for any further appropriate testing.
(2) If toxin production by the MPCA is suspected, or if toxin
production is indicated by significant or persistent signs of toxicity
in the test animals, in the absence of signs of infectivity or
pathogenicity, then, (i) the toxic component (s) of the dosing
material is (are) to be identified, and to a practical extent,
isolated.
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Subdivision M
(ii) An acute toxicity study (152A-20) is to be conducted with
the toxic component(s).
(3) If significant infectivity or unusual persistence of the
MPCA is observed in the absence of signs of toxicity or pathogenicity,
then a subchronic study ( 152A-21) is required.
152A-13 Acute intravenous toxicity/pathogenicity study with
microbial pest control agents f(MPCA)s'); Tier I.
(a) When required. Acute intravenous toxicity/pathogenicity
data are required by 40 CFR 158.740 to support the registration
of each manufacturing-use product and each end-use product, and the
technical grade of each active ingredient.
(b) Purpose. Acute intravenous toxicity/pathogenicity data
provide information.on health hazards likely to arise from a single
exposure where the skin is bypassed as a barrier. The purpose of
the acute intravenous study is to provide initial information on
the toxicity, infectivity, and pathogenicity of a MPCA. using a
single high dose exposure and an adequate post-exposure observation
period.
(c) Definitions.
(1) "Acute intravenous toxicity" and "acute intravenous
pathogenicity" are the adverse effects occurring from the intravenous
administration of a MPCA Acute intravenous toxicity also may be the
adverse effects occurring from the intravenous administration of a
microbially produced substance.
(2) "Dose level" is the amount of MPCA. administered. It is
expressed as units of the microorganism administered per test animal.
(3) "Units of MPCAs". One unit of representative MPCA groups
usually would be defined as follows:
(i) vegetative bacterium: a single, viable organism, and
usually the entity that produces a single colony forming unit (CPU) on
an appropriate semisolid growth medium.
(ii) bacterial or fungal spore, bacterial or protozoan cyst: an
intact individual spore or cyst as determined microscopically, and
usually the entity that produces a single CPU on appropriate germination
medium.
(iii) fungal mycelium: 10~9 gram dry weight or, after standardized
preparatory procedures, a inycelium-producing entity on semi-solid growth
medium.
(iv) virus: an intact, complete virion or a polyhedral body as
determined by electron microscopy, and, usually the entity that produces
an infective unit (IU) on appropriate host cells or tissues.
(v) protozoa: an intact vegetative organism, spore, or cyst of
the members in the various classes of this phylum.
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Pesticide Assessment Guidelines
IXie to the wide diversity of forms among microorganisms, other
definitions of a unit of a MPCA may be equally appropriate.
(d) Principles of the test method. Ihe test MPCA is administered
by intravenous injection in a single high dose to experimental animals.
Subsequent observations of effects and deaths are made, and the rate of
clearance of the MPCA is estimated. Animals that die during the test
are necropsied, and at the conclusion of the test the surviving animals
are sacrificed and necropsied. Infectivity of the MPCA is evaluated
periodically during the test and at the conclusion of the test.
(e) Substance to be tested. (1) The technical grade of each
active ingredient shall be tested to support the registration of each
manufacturing-use product and each end-use product.
(2) Ihe physical nature of some technical grade preparations
may render intravenous testing impractical. In such cases, the test
substance may consist only of the purest infective form of the micro-
organism. However, justification/reasoning must be provided by the
investigator to support a request for exempting specific technical
grade preparations from testing by intravenous dosing.
(3) The form (e.g. vegetative cell, spore, cyst, virion) of
the MPCA used in testing should be equivalent to the form that is
intended for registration or application. To the extent possible,
the test MPCA also should be equivalent to the MPCA intended for
registration or application with respect to stage of growth, posses-
sion of organelles and appendages and expression of phenotypic
traits (including products from intentionally introduced genes).
If significant exposure to other forms of the MPCA are expected, or
if changes in form of the MPCA occur in the host, then these forms
also may have to be tested.
(f) Characteristics of the test MPCA. The test MPCA should
be thoroughly characterized as required in section 151A of these
guidelines.
(g) Test procedures.
(1) Animal selection.
(i) Species and strain. Although several mammalian test
species may be used, the mouse or the rat are the preferred rodent
species. Commonly used laboratory strains should be used. If
another species is used, justification/reasoning for the alternative
selection should be provided. All test animals should be free of
parasites or pathogens. Females should be nulliparous and non-preg-
nant.
(ii) Age. Young adult animals should be used . The weight
variation of animals used in a test should not exceed ± 20 percent
of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex are required.
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Subdivision M
(iv) Numbers. At least 6 animals (three animals of each sex)
should be used. A sufficient number of additional animals should
be used to allow for interim sacrifice for infectivity determinations
and also for control groups.
(2) Control groups.
(i) A concurrent untreated control group of two animals/sex is
required. A "shelf control group" is not required.
(ii) A separate vehicle control group is not required except
when the toxicity of the vehicle is unknown.
(iii) Control groups dosed with inactivated MPCA (i.e. ren-
dered incapable of reproduction or germination or excystment) may
prove useful to evaluate toxic properties of the MPCA. If included,
inactivation should be done by a means that allows for reasonable
maintenance of the structural integrity of the MPCA.
(3) Dosing.
(i) Dose level. One dose level of at least 107 units of the
MPCA per test animal should be used. If a dose level of at least
107 units per test animal, a justification/explanation must be
provided.
(ii) Vehicle. The recommended vehicle is one that allows for
maintenance of viability, or germination capability, or excystanent
capability, or, for intracellular parasites, infection capability
in a suitable host.
(iii) Volume. The maximum volume of liquid that can be adminis-
tered by intravenous injection at one time depends on the size of
the test animal. Variability in test volume should be minimized.
(iv) Dose quantification. Techniques used to quantify the
units of MPCA in any dose will depend on the group of microorganisms
to which the MPCA belongs. Where possible, determinations of viable,
or potentially viable, or infective units in each dose should be
made. A measurement of metabolism associated with a defined biomass
may be the preferred technique for quantification of mycelial forms
of MPCAs. Quantification should be done concurrently with testing.
(4) Exposure.
(i) The test substance should be administered intravenously
via a needle and syringe in a single dose.
(ii) If a single dose is not possible, the dose may be given
in smaller fractions over a period not exceeding 24 hours.
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Pesticide Assessment Guidelines
(5) Observation period, The observation period should be at
least for 21 days after dosing. However, the duration of observation
should not be fixed rigidly. It should be determined by the type
of MPCA administered and its rate of clearance from the test animals.
IXiration of the observation period also would depend on the time at
which signs of toxicity and pathogenicity appear and disappear, and
the time to death of the animals.
(6) Observation of animals.
(i) A careful clinical examination of all animals should be
made at least once each day.
(ii) Additional observations should be made daily with appro-
priate actions taken to minimize loss of animals to the study,
e.g., necropsy of, and MPCA enumeration from those animals found
dead, and isolation of weak or moribund animals.
(iii) Cageside observations should include, but not be limited
to, changes in :
(A) the skin and fur;
(B) eyes and mucous membranes;
(C) respiratory system;
(D) circulatory system;
(E) autonomic and central nervous system;
(F) somatomotor activity/ and,
(G) behavior pattern.
(H) Particular attention should be directed to observation of
tremors, convulsions, diarrhea, lethargy, salivation, sleep and
coma.
(iv) Individual weights of animals should be determined shortly
before the test material is administered, weekly thereafter, and at
death or at interim or final sacrifice. Changes in weight should
be calculated and recorded when survival exceeds one day.
(v) The time of death should be recorded as precisely as possible.
(7) Gross pathology. A gross necropsy of all animals should
be performed. All gross pathological changes should be recorded.
(8) Clearance of MPCA. Blood from test animals should be
collected during the study and examined for the presence of the
MPCA to estimate clearance of the MPCA after dosing. The first
analysis should be done on three animals/sex as soon as reasonably
possible after dosing. Methods (e.g. immunological assays, ENA
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Subdivision M
probes) other than those used for quantification of MPCAs in each dose
may prove useful. Recovery values and detection and sensitivity limits
should be determined and reported for each technique used.
(9) MPCA enumeration in tissues, organs, and body fluids.
Infectivity and clearance should be assessed by using sensitive techniques
to determine the presence of the MPCA in tissues, organs and body fluids.
Methods other than those used for quantification of MPCAs in each dose
may prove useful. Recovery values and detection and sensitivity limits
should be determined and reported for each technique used. Methods
selected for MPCA enumeration should, if possible, allow for detection
of microbial replication.
(10) Interim sacrifice. For evaluating infectivity and clearance, the
MPCA should be enumerated from tissues, organs, and body fluids of three
treated animals per sex, sacrificed at three days after, and then at one
week intervals after dosing. The number of interim sacrifice periods re-
quired will depend on the nature of the test microorganism, and should be
sufficient to adequately establish a pattern of clearance.
(11) Tissues, organs, and body fluids.
(i) For infectivity and persistence determinations, the MPCA
should be enumerated from the kidneys, brain, liver, lung, spleen,
blood, representative lymph nodes, caecum contents, and, where appro-
priate, from lesions and the inoculation site.
(ii) Other tissues, organs, and body fluids may have to be examined
as indicated by the nature of any toxic and pathogenic effects observed.
(i) Data and reporting.
(1) Treatment of results. In addition to the information recommended
by 150A-4, the test report should include the following information:
(i) number of animals at the start of the test;
(ii) time of death of individual animals;
(iii) number of animals displaying other signs of toxicity
and pathogenicity;
(iv) description of toxic and pathogenic effects;
(v) definition for one unit of the MPCA used, and the units/test
animal in the dose;
(vi) body weights and time taken;
(vii) necropsy findings;
(viii) pathology findings, when performed;
(ix) infectivity findings;
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pesticide Assessment Guidelines
(x) estimate of rate of MPCA clearance;
(xi) description of all enumeration methods used for MPCA
detection and quantification, and
(xii) verification that each enumeration method is sufficiently
sensitive to serve as a useful quantitative assay for the MPCA in
tissues, organs, and body fluids.
(2) Evaluation of results. An evaluation should include the
relationship, if any, between exposure to the test substance and
the incidence and severity of all abnormalities, including;
(i) behavioral abnormalities;
(ii) clinical abnormalities;
(iii) gross lesions;
(iv) body weight changes;
(v) mortality;
(vi) toxicity;
(vii) infectivity; and
(viii) pathogenicity.
(j) Tier progression
(1) If persistent or significant signs of pathogenicity of the
MPCA for the test animals is observed in Tier I, acute intravenous
toxicity/pathogenicity tests may be required in non-rodent animal
species. Consultation with the Agency is recommended to determine
the requirement for any further appropriate testing.
(2) If toxin production by the MPCA is suspected, or if toxin
production is indicated by significant or persistent signs of toxicity
in the test animals, in the absence of signs of infectivity or
pathogenicity, then;
(i) The toxic component (s) of the dosing material are to be
identified, and where possible, isolated.
(ii) An acute toxicity study ( 152A-20) is to be conducted
with the toxic component (s).
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Subdivision M
152A-14 Primary eve irritation study with microbial
pest control agents rfMPCZQsl; Tier I.
(a) When required. Primary eye irritation data are required
by 40 CER 158.740 to support the registration of each manufacturing-
use product and each end-use product.
(b) Purpose. Primary eye irritation data provide information
on possible hazards associated with a single application of the
MPCA to the eye, and/or associated with inert ingredients in formu-
lations of the MPCA, and/or associated with products from genes
intentionally introduced into the MPCA.
(c) Definitions.
(1) "Eye irritation" is the production of reversible changes
in the eye following the application of a test substance to the
anterior surface of the eye.
(2) "Eye corrosion" is the production of irreversible changes
in the eye following the application of a test substance to the
anterior surface of the eye.
(3) "Dose level" is based on the amount of MPCA administered.
It is expressed as;
(i) units of the microorganism administered at each test site
on the test animal, and, if applicable, as:
(ii) dry weight of applied test substance at each test site
per kilogram body weight of test animal.
(4) "Units of MPCAs". One unit of representative MPCA groups
usually would be defined as follows:
(i) vegetative bacterium: a single, viable organism, and
usually the entity that produces a single colony forming unit (CTU)
on an appropriate semisolid growth medium.
(ii) bacterial or fungal spore, bacterial or protozoan cyst:
an intact individual spore or cyst as determined microscopically,
and usually the entity that produces a single CFU on appropriate
germination medium.
(iii) fungal mycelium: 10~9 gram dry weight or, after stan-
dardized preparatory procedures, a mycelium-producing entity on
semi-solid growth medium.
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Pesticide Assessment Guidelines
(iv) virus: an intact, couplets virion or a polyhedral body-
as determined by electron microscopy, and, usually the entity that
produces an infective unit (IU) on appropriate host cells or tissues.
(v) protozoa: an intact vegetative organism, spore, or cyst
of the members in the various classes of this phylum.
Due to the wide diversity of forms among microorganisms, other
definitions of a unit of MPCA may be equally appropriate.
(d) Principles of the test method. Each formulation of the
MPCA. to be tested is applied in a single dose to one eye of each
experimental animal, with the untreated eye of each animal serving
as a control. The degree of irritation is read and scored at specified
intervals and is further described to provide a complete evaluation
of the effects.
(e) Substance to be tested.
(1) The manufacturing-use product shall be tested to support
the registration of each manufacturing-use product.
(2) The end-use product shall be tested to support the regis-
tration of each end-use product.
(3) The form (e.g. vegetative cell, spore, cyst, virion) of
the MPCA used for preparation of the dosing material should be
equivalent to the form that is intended for registration or application.
To the extent possible, the test MPCA also should be equivalent to
the MPCA intended for registration or application with respect to
stage of growth, possession of organelles and appendages, and expression
of phenotypic traits (including products from intentionally introduced
genes). If significant exposure to other forms of the MPCA are
expected, then these forms also may have to be tested.
(4) If the test formulation contains substances of known
corrosive properties, which, for example, have produced definite
corrosion or severe irritation in a dermal study, then it need not
be tested for eye irritation or infection.
(f) Characteristics of the test MPCA. The test MPCA should
be thoroughly characterized as required in section 151A of these
guidelines.
(g) Test procedures.
(1) Animal selection.
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Subdivision M
(i) Species and strain. Although several mammalian test species
may be used, the albino rabbit is the preferred species. Commonly
used laboratory strains should be used. If another species is
used, the tester should provide justification/reasoning for the
alternative selection. All test animals should be free of parasites
or pathogens. Females should be nulliparous
and non-pregnant.
(ii) Number and age of animals. At least six adult animals
should be used, unless reasonable justification for using fewer
animals is provided.
(2) Control groups.
(i) Separate animals are not needed for an untreated control
group, since each test animal serves as its own control.
(ii) A vehicle control group is recommended if the vehicle
is known to cause any toxic eye reactions, or if the ocular effects
of the vehicle are unknown.
(3) Dosing.
(i) MPCA level. One dose level of the manufacturing-use pro-
duct containing at least 107 units of the MPCA should be applied
to each test eye. If a dose level of at least 107 units of MPCA
is not used, a justification/explanation must be provided. The
units of MPCA in the dose for the end-use product are enumerated
concurrent with testing.
(ii) Vehicle. The recommended vehicle for the technical grade
of the active ingredient is one that allows maintenance of viability,
or germination capability/ or excystment capability, or, for intra-
cellular parasites, infection capability in a suitable host. The
recommended vehicle for the manufacturing-use product or end-use
product is the same material in which the MPCA will be distributed,
mixed, suspended, or diluted for application.
(iii) Dose level. A volume of 0.1 ml is used where the product
to be tested is an MPCA suspension largely comprised of a liquid
phase. When the product to be tested is a solid or a slurry, then
0.1 g should be used unless justification for use of other weights
of the test substance is provided. The units of MPCA in the dose
of each test product must be quantified concurrent with testing.
(iv) Dose quantification. Techniques used to quantify the
units of MPCA in any dose will depend on the group of itd<3roorganisms
to which the MPCA belongs. Where possible, determinations of viable,
or potentially viable, or infective units in each dose should be
made. A measurement of metabolism associated with a defined biomass
may be the preferred technique for quantification of mycelial MPCAs.
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Pesticide Assessment Guidelines
(4) Examination of eves prior to test. Both eyes of each
experimental animal provisionally selected for testing should be
examined within 24 hours before testing starts. Any animal showing
eye irritation or infection, ocular defects, or pre-existing corneal
injury should not be used.
(5) Application of the test substance.
(i) The test substance should be placed in the conjunctiva!
sac of one eye of each animal after gently pulling the lower lid
away from the eyeball. The lids are then held together for about
one second to prevent loss of the material. The contralateral,
untreated eye serves as a control. A local anaesthetic to reduce
pain may be used prior to instillation of the test substance, provided
that evidence can be presented to show that the anaesthetic does
not influence the reaction to the test substance. The control eye
also should be anaesthetized.
(ii) The eyes of the test animals should not be washed out
until 24 hours after instillation of the test substance. At this
time, lukewarm water may be used to wash the eyes.
(6) Observation period. The duration of the observation period
should not be fixed rigidly, but it should be sufficient to evaluate
fully the reversibility or irreversibility of the effects observed.
It normally not exceed 21 days after instillation.
(7) Clinical examination and scoring, (i) All eyes should
be examined for ocular lesions at 1, 24, 48, and 72 hours and at 4
and 7 days after treatment. If irritation is observed at 7 days,
then examinations should be made every 3 days thereafter for 21
days after treatment. If there is no evidence of irritation at 7
days after treatment, the study may be ended. In addition to the
observations of the cornea, iris, and cxanjunctivae, any other observed
lesions should be reported. Periocular regions also should be examined
for possible lesions. Grading and scoring of ocular lesions are to
be done using in accordance with Table 2 (from Draize, et al.,
1965).
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Subdivision M
TABLE 2: GRADES FOR OCUIAR LESIONS
CORNEA
(A) Opacity; degree of density (most dense area is taken for reading)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of
normal luster), details of iris clearly visible 1
Easily discernible translucent area, details of iris slightly obscured 2
Nacrous area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4
(B) Area of cornea involved
None 0
One-quarter (or less) but not zero 1
Greater than one-quarter, but less than one-half 2
Greater than one-half, but less than three-quarters 3
Greater than three-quarters 4
Score equals A x B x 5 Total possible maximum = 80
IRIS
(A) Values
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal
hyperemia, or injection, (any of these or combination thereof), iris
still reacting to light (sluggish reaction is positive) 1
No reaction to light, hemorrhage, gross destruction (any or all of these) 2
Score equals A x 5 Total possible maximum = 10
O3NJUNCITVAE
(A) Redness (refers to palpebral and bulbar conjunctivae, cornea, and iris)
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse crimson color, individual vessels not easily discernible 2
Diffuse beefy red 3
(B) Chemosis
No swelling 0
Any swelling above normal (includes nictitating membrane) 1
Obvious swelling with partial aversion of lids 2
Swelling with lids about half closed 3
Swelling with lids from half to completely closed 4
(C) Discharge
No discharge ....0
Any amount different from normal (does not include small amounts
observed in inner canthus of normal animals) 1
Discharge with moistening of the lids and hairs just adjacent to lids).. .2
Discharge with moistening of the lids and hairs, and considerable
area around the eye 3
Score equals (A + B + C) x 2 Total possible maximum = 20
Total possible maximum score is the sum of all scores obtained for the
cornea, iris, and conjuctivae.
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pesticide Assessment Guidelines
(ii) Examination of reactions can be facilitated by use of a
binocular loupe, hand-slit lamp, biomicroscope, or other suitable
device. After recording the observations at 24 hours, the eyes of
any or all rabbits may be further examined with fluorescein.
(h) Data and reporting.
(1) Data shall be presented in tabular form, showing, for
each individual animal:
(i) The irritation scores at the designated observation time;
(ii) A description of the degree and nature of irritation;
(iii) The presence of serious lesions; and
(iv) Any effects other than ocular which were observed.
(2) Evaluation of the results. The eye irritation scores shall
be evaluated in conjunction with the nature and reversibility, or
otherwise, of the responses observed. The individual scores do not
represent an absolute standard for the irritant properties of a
material. They shall be viewed as reference values and are only
meaningful when supported by a full description and evaluation of
the observations.
(3) Test report. In addition to the information required by
150A-4, the test report shall include the following information:
(i) description of the physical nature of the test substance;
(ii) units of MPCA in the dose of each formulation tested,
and, where appropriate, the weight of the test substance in each
dose;
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Subdivision M
(iii) species and strain of test animal used;
(iv) narrative description of the degree and nature of irrita-
tion or corrosion or lesions or infections observed;
(v) description of any non-ocular effects observed;
(vi) description of the method used to score the irritation
(e.g. hand slit-lamp, bicmicroscope, fluorescein); and
(i) Tier progression. No further testing is required.
152A-15 Hypersensitivitv incidents with microbial pest control
agents FfMPCA^sl; Tier I
(a) When required. Data on incidents of hypersensitivity,
including immediate-type and delayed-type reactions, of humans or
domestic animals that occur during the production or testing of the
technical grade of the active ingredient, the manufacturing-use
product, or the end-use product should be reported in support of an
application for registration. For reporting of incidents taking
place after registration, refer to the requirements in connection
with sec. 6(a) (2) of FIFRA.
(b) Reporting. The reporting requirements for these incidents
should be the same as those for conventional chemical pesticide
incident reports as specified in the Pesticide Incident Report form
(EPA form number 8550-5, CMB form number 158-R0008). The following
information should be provided, if available:
(1) A description of the ingredients (including the MPCA) to
which the affected individual (s) were exposed;
(2) The frequency and duration of exposure to the material,
and route(s) of exposure;
(3) The time, date, and location of exposure to the material;
(4) The situation or circumstances under which exposure to the
material occurred; and,
(5) any clinical observations.
,-, r—^ression. If any incidents of hypersensitivity of
humans or domestic animals are observed during the production or
testing of the technical grade of the active ingredient, the manu-
facturinguse product, or the end-use product, then Tier II testing
is required. The specific Tier II test requirements will depend on
the nature of the observations and will be determined after consul-
tation with the Agency.
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pesticide Assessment Guidelines
152A-15
(a) When required. Data from cell culture tests with viruses
are required by 40 CER 158.740 to support the registration of each
manufacturing-use product and each end-use product. See 40 CER
158.50 and 158.740 to determine whether these data are to be
submitted.
(b) Purpose. Cell culture tests provide information on the
ability of viral pest control agents to infect, replicate in, transform,
or cause toxicity in, mammalian cell lines.
(c) Definitions
(1) Most infectious form (MIF): The form or preparation of
virus that gives optimal infection in the susceptible cell culture
or organism. For non-occluded viruses, the MIF is the purified
virus or purified tissues obtained from an infected host. For
occluded viruses (e.g. baculoviruses, cytqplasmic polyhedrosis
viruses, entomopox viruses) the MIF for cell culture or injection
into an organism is extracellular virus found in cell culture
medium or in infectious hemolymph. Ihe MIF for susceptible insect
hosts for infection by natural routes (feeding) is the viral occlu-
sion body.
(2) Viral Toxicity: Ihe ability of a virus to inflict injury
or damage to a host cell, where infection by, and/or replication of
the virus are not necessarily required. Toxicity can also be the
ability of non-viral components of a preparation to inflict injury
or damage to a host cell.
(3) Viral Infectivity: The ability of viral genes to become
established in a host cell genome, or the ability of viral genes
to be expressed in a host cell (resulting in the production of
virus-encoded nucleic acids).
(4) Transformation: The detectable modification of a host
cell phenotype induced by the presence of viral nucleic acid.
Transformed cells are considered to be infected by the virus.
(5) Cytopathic effects (CPE): Any host cell damage or injury
resulting from infection of the cell by a virus. These effects can
be morphological or biochemical, and include but are not limited
to, cell growth, attachment, morphology, nucleus size and shape,
and cellular processes such as macromolecular synthesis.
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Subdivision M
(d) Test standards.
(1) Substance to be tested. Hie purest, most infectious
form (MIF) of the virus should be used. Preparations of insect viruses
should be free of insect hemolymph, unless it has been determined that
the hemolymph is not toxic to the cell cultures used. The inoculum
should be titered by the most sensitive assay available, and in the
most permissive host system (cell culture or, if not available, host
organism). For testing in the model systems, a minimum of five
plaque-forming units (PFU) per cell is required when a plaque assay for
the virus is available, or seven times the ID^Q units when a plaque
assay for the virus is not available. If fewer units per cell or organism
are used, then justification/reasoning must be provided for using the
lesser amount.
(2) Cell cultures. The following cells are recommended: one
human line (e.g., WI38); one primary cell type (e.g., foreskin); one
primate continuous line (e.g., monkey CV-1 or BSC-1); primary Syrian
hamster embryo (SHE) cells (to provide data for the cell transformation
assay, described below). One other cell line is to be selected to
evaluate potential concerns intrinsic to the specific viral pest control
agent, and its intended use. Justification/reasoning must be provided
for the selection of this latter cell line.
(3) Toxicitv evaluation. Efficiency-of-plating tests should be
performed with each cell line. For each cell line, approximately two
hundred cells are plated on each of 30 dishes. At 24 hours post-plating,
10 dishes/cell line are exposed to approximately 106 units of the
virus. Appropriate vertebrate cell culture medium is added to 10 different
dishes, and, if applicable, 10 dishes/cell line are exposed to invertebrate
medium only. At 1 hour post-exposure, all cultures are fed with the
appropriate vertebrate cell culture medium, and then are incubated
until control cultures have colonies consisting of at least 25 cells/
colony. All cultures then are fixed and stained, and colonies are
enumerated.
(4) Infectivity evaluation.
(i) Subconfluent cultures (containing approximately 2xl05 cells
on 25 cm2 dishes) of each cell line are exposed to >lx!06 units of
the viral pest control agent. Appropriate controls include subconfluent
cultures that receive no treatment, and those that are exposed to virus-
free inoculation medium. At 7 days and at 14 days post-inoculation,
cells are to be subcultured.
(ii) Cell cultures are observed daily for 21 days post-inoculation
for appearance of CPE.
(iii) Cultures are quantitatively assayed for the virus on days
1, 2, 5, 7, 14, and 21 post-inoculation.
(A) Cells (entire culture, or >2xl05 cells) are to be assayed in
triplicate for viral antigen and nucleic acid.
(B) Cell culture fluid from replicate cultures is to be assayed
for infectious virus, using an appropriate susceptible host model system.
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Pesticide Assessment Guidelines
(iv) Assays for fate of input virus and for presence of viral
protein(s) and nucleic acid.
(A) The enzyme linked immunosorbent assay (ELISA), dot-immuno-
binding assay (Tijssen, 1985; Hawkes et al., 1982; Kara et al., 1982),
protein blot immunoassay (Western transfer) (Smith and Summers, 1981;
Volkman and Goldsmith, 1982) or similar assays are recommended for
protein determination.
(B) The dot hybridization procedure (Bishop, 1983; Kafatos, et al.,
1979; Brandsma and Miller, 1980), Southern hybridization procedure
(Southern, 1975; Smith and Summers, 1983), or other similar assay using
probes of high specific activity are recommended for nucleic acid deter-
minations.
(v) Controls.
(A) For each cell culture, cells inoculated with a preparation of
the inactivated test virus should be analyzed as described for the
active test virus.
(B) For each series of tests, the inoculum should be tested in
the permissive cell line or host organism as a positive control and for
direct reference to the data obtained from the vertebrate cell lines.
(5) Cell transformation assay.
(i) The ability of the viral pest control agent to transform primary
Syrian hamster embryo (SHE) cells is to be determined, using an appro-
priate assay system. If other test systems are used, then justification/
rationale must be presented to show that the alternate systems are appro-
priate.
(ii) Controls.
(A) Transformation of SHE cells with Simian adenovirus 7 (SAV 7)
serves as the positive control.
(B) SHE cells treated with cell culture medium alone, and SHE cells
treated with a killed preparation of the inactivated viral pest control
agent serve as appropriate negative controls. The inactivation procedure
must be demonstrated as effective in preventing transformation.
(C) An efficiency of plating test with SHE cells (see above) is
considered an appropriate toxicity control.
(iii) If the data show that the test virus modifies the cell phe-
notype, then cells from cultures derived from morphologically trans-
formed colonies are to be inoculated into hamsters, and tumorigenesis
in the host animal is to be evaluated.
(iv) This assay may not be required if, in the infectivity evalua-
tions (d (4), above), it is conclusively demonstrated that viral nucleic
acid is not persistent in any of the test cell lines employed.
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Subdivision M
(c) Data reporting and evaluation. The following information
should be provided for each test:
(1) CPE in the cell monolavers.
(i) The appearance of CPE should be described in such a way that
virus-induced cell destruction is differentiated from non-specific
effects.
(ii) Cultures should be inspected with the aid of a microscope
to provide evidence of CPE that should be recorded as:
1+ = suggestive of virus-induced morphologic changes
2+ = definitive morphologic changes
3+ = more than 50% cell degeneration; or
4+ = complete cell destruction
(iii) The TCIDgQ value calculated by an appropriate statistical
method. For computation of the infectiyity results, only cultures
showing a >2+ CPE are considered to be infected.
(2) Toxicitv evaluation.
(i) Details of all procedures used, including appropriate reagents
and materials, and assay sensitivities and limitations.
(ii) Efficiency of plating data of cultures receiving virus, and
cultures receiving vertebrate media (control cultures) and invertebrate
media.
(iii) Assessment of mitotic process prevention or of interference
with chromosomal replication, as indicated for example, by significant
reductions in efficiency of plating.
(3) Assay of culture fluid.
(i) Details of procedures used, including a discussion of all
data that indicate viral replication.
(4) Data from assays of input viruses.
(i) Details of procedures used for detection of viral antigens and
nucleic acids and their persistence in culture, including appropriate
reagents and materials, and assay sensitivities and limitations.
(ii) Intracellular concentration of viral antigens and viral
nucleic acids, reported as a function of cell number (e.g., viral genome
number/cell).
(5) Cell transformation assay.
(i) Details of the protocols used for the cell transformation assay,
and reference to tha assay used, if published.
(ii) Control value data, including efficiency of plating results.
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Pesticide Assessment Guidelines
(iii) Tumorogenesis data in test animals, if this study is required.
(6) General information to be provided for all tests:
(i) The source of each cell line used;
(ii) Evidence for lack of adventitious agents in cell lines;
(iii) Information on genetic stability of continuous cell lines,
and on donors of primary cells.
(d) Tier progression. The requirement for further studies as
indicated by the data from these studies also will be based on data
from the other test requirements in this Tier.
(1) If the data show that the viral pest control agent is not
cytotoxic, nor does it infect, replicate in, or transform any cell
culture, then no further testing is required.
(2) If the data show that the viral pest control agent preparation
is toxic to any of the test cell cultures, but does not infect, replicate
in, or transform any of the cell cultures, then: (i) the toxic component(s)
of the preparation may have to be identified; and,
(ii) an acute toxicity study ( 152A-20) may be required with the
toxic component(s).
(3) If the viral pest control agent infects any of the test cell
cultures, then reproductive and fertility effects ( 152A-30), oncogenicity
( 152A-31), immunodeficiency ( 152A-32), and primate infectivity/path-
ogenicity ( 152A-33) studies may be required.
(e) References.
(1) Bishop, D. H. L. (1983) The application of RNA finger
printing and sequencing to viral diagnosis. Curr. Topics Microbiol.
Immunol. 104:259-271.
(2) Brandsma, J. and G. Miller (1980) Nucleic acid spot
hybridization: rapid quantitative screening of lymphoid cell lines
for Epstein-Barr viral ENA. Proc. Nat'l. Acad. Sci. USA 77:6851-6855.
(3) Casto, B.C. (1968) Adenovirus transformation of hamster
embryo cells. J. Virol. 2:376-383.
(4) Hames, B.D. and S.J. Higgens (eds) (1985) Nucleic acid
hybridisation. A practical approach. IRL Press, Washington, D.C.
(ISBN 0-947946-23-3).
(5) Heidelberberger, et al. (1983) Cell transformation by
chemical agents -a review and analysis of the literature. A report
of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation
Res. 114:283-385.
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Subdivision M
(6) Kafatos, F.C., C.W. Jones, and A. Efstratiadis (1979)
Determination of nucleic acid sequence homologies and relative
concentrations by a dot hybridization procedure. Nucleic Acids Res.
7:1541-1552.
(7) Smith, G. and M.D. Summers (1981) Application of a novel
radioimmunoassay to identify baculovirus structural proteins that
share interspecies antigenic determinants. J. Virology 39:125-137,
(8) Southern, E. (1975) Detection of specific sequences among
DNA fragments separated by gel electrophoresis. J. Mbl. Biol.
98:503.
(9) Tijssen, P. (1985) Laboratory techniques in biochemistry
and molecular biology. Practice and theory of enzyme immunoassays.
Elsevier Science Publishing (ISBN 0-444-80633-4)
152A-17 [Reserved]
152A-18 [Reserved]
152A-19 [Reserved]
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Pesticide Assessment Guidelines
Group 2: Tier II Testing.
152A-20 Acute toxicity study with itiicrdbial
F (MPCA)si; Tier II
(a) When required. Acute toxicity data are required by
40 CFR 158.740 to support the registration of each manufacturing-use
product and each end-use project and the technical grade of each
active ingredient when significant or persistent signs of toxicity
are observed in test animals from Tier I studies, in the absence of
signs of significant infectivity or pathogenicity.
(b) Purpose. Acute toxicity data provide information on health
hazards likely to arise from a single exposure to toxins or toxic
components derived from, or associated with the test substance.
The toxic components are to be isolated and identified. The purpose
of an acute toxicity study is to determine the median lethal dose,
its statistical limits, and slope using a single exposure
and a 14-day post-exposure observation period.
(c) Definitions.
(1) "Acute toxicity" is the adverse effects occurring from
administration of a single dose of a component, or components, of
the test substance.
(2) "Median lethal dose" is a statistically derived single
dose of a substance that can be expected to cause death in 50 percent
of animals. It is expressed in terms of weight of test substance
per unit weight of test animal, and also in terms of the proportion,
by weight, of the toxic component (s) in the test substance per unit
weight of test animal.
(d) Principle of the test method. The test substance, including
toxic components, is administered in graduated doses to several
groups of experimental animals, one dose being used per group.
Subsequent observations of effects and deaths are made. Animals
which die during the test are necropsied, and at the conclusion of
the test, the surviving animals are sacrificed and necropsied as
indicated by the nature of the toxic effects observed.
(e) Substance to be tested. The toxic component (s) of the MPCA
preparation are to be isolated and identified. The test substance
will comprise an appropriately purified preparation of the toxic
components. The proportion by weight of the toxic components in
the test substance is to be determined and reported.
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Subdivision M
(f) Test procedures. (1) Animal selection. The species and
strains of test animal to be used are those in which toxic effects
were observed in the acute toxicity/ pathogenicity studies from
Tier I.
(2) Route of exposure. The route or routes of exposure should
correspond to each and all routes (i.e., oral, dermal, and pulmonary)
where toxicity was observed in the acute toxicity/pathogenicity
studies from Tier I. A separate acute toxicity test is required
for each route of exposure.
(3) Other test standards, (i) The applicable test standards
set forth in 152A-10, 152A-11, and 152A-12, and of this Sub-
division, and in the corresponding tests in Subdivision F (i.e.,
81-1, 81-2, and 81-3) should be followed.
(ii) If acute pulmonary toxicity tests are required, then
the applicable standards set forth in 152A-12 of this Subdivision
usually will take precedence over the standards set forth in 81-3
of Subdivision F.
(g) Tier progression. The Agency will make recommendations
on appropriate further test requirements. In general, further test
requirements will be comprised of appropriate tests as described
for biochemical pest control agents in this Subdivision or for
chemical pesticides as described in Subdivision F.
152A-21. Subchronic toxicity/pathogenicity studies with microbial
pest control agents [(MPCA)s]t Tier II.
(a) When required. Subchronic toxicity/pathogenicity studies
are required by 40 CFR 158.740 to support the registration of
each manufacturing-use product and each end-use product and the
technical grade of each active ingredient when significant infectivity
and/or unusual persistence of the MPCA is observed in test animals
from the Tier I studies in the absence of significant pathogenicity
or toxicity. These studies also may be required to provide an
evaluation of adverse effects due to microbial contaminants or toxic
by-products in a MPCA preparation.
(b) Purpose. Subchronic toxicity/pathogenicity data provide
information on health hazards likely to arise from subchronic (90-day)
exposure to a MPCA preparation.
(c) Principle of the test method. The test substance is adminis-
tered daily to experimental animals at a single high dose level for
a time period of at least 90 consecutive days. During this period,
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animals are observed daily to detect signs of toxicity and patho-
genicity- Animals which die during the test period are necropsied,
and the MPCA is enumerated from appropriate tissues, organs, and
body fluids. At the conclusion of the test, surviving animals are
sacrificed and necropsied, and appropriate tissues, organs, and
body fluids are analyzed for the quantitative presence of the MPCA.
(d) Substance to be tested.
(1) The manufacturing-use product and, if different, the
technical grade of each active ingredient shall be tested to support
the registration of a manufacturing-use product.
(2) Ihe end-use product shall be tested to support the regis-
tration of an end-use product.
(3) Usually, the form of the MPCA to be tested will be equi-
valent to the form used in the acute toxicity/pathogenicity Tier I
studies of this Subdivision, and which resulted in significant
signs of inf ectivity or unusual persistence without accompanying
signs of pathogenicity or toxicity.
(e) Test procedures.
(1) Animal selection.
(i) Species and strain. The species and strains of test animal
to be used are those in which infectivity/unusual persistence of
the MPCA was observed in the acute toxicity/pathogenicity Tier I
studies, and in which no significant signs of pathogenicity or
toxicity were observed. All test animals should be free of parasites
or pathogens. Females should be nulliparous and non-pregnant.
(ii) Age. Young adult animals should be used. The weight
variation of animals used should not exceed ± 20 percent of the
mean weight for each sex.
(iii) Sex and numbers. At least twenty animals (ten animals
of each sex) should be treated with the MPCA.
(2) Control groups.
(i) A concurrent untreated control group is required.
(ii) A separate vehicle control group is not required except
when the toxicity of the vehicle is unknown.
(iii) A control group dosed with inactivated MPCA (i.e.,
rendered incapable of reproduction or germination or excystment) may
prove useful to evaluate toxic properties of the MPCA. Uiactivation
should be done by a means that allows for reasonable maintenance of
the structural integrity of the MPCA.
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Subdivision M
(3) Dosing.
(i) Dose level. A dose level of >108 units of viable
MPCA per test animal is to be administered daily to each test animal.
If a dose level of at least 108 units of MPCA per test animal is
not used, a justification/explanation must be provided.
(ii) Vehicle. The recommended vehicle for the technical grade
for each active ingredient is one that allows for maintenance of
viability, or germination capability, or excystment capability, or,
for intracellular parasites, infection capability in a suitable
host. The recommended vehicle for the manufacturing-use product or
end-use product is the same material in which the MPCA will be
distributed, mixed, suspended, or diluted for application.
(iii) Route of exposure. The oral route is to be used if
significant infectivity/ unusual persistence of the MPCA was observed
in test animals in the acute oral toxicity/pathogenicity Tier I
study ( 152A-10). The inhalation route (usually intranasal instil-
lation) is to be used if significant infectivity/unusual persistence
of the MPCA was observed in test animals in the acute pulmonary
toxicity/pathogenicity Tier I study ( 152A-12).
(4) Observation of animals.
(i) A careful cageside examination of each test animal should
be made at least once per day.
(ii) Additional observations should be made daily with appro-
priate actions taken to minimize loss of animals to the study,
e.g., necropsy of, and MPCA enumeration from those animals found
dead, and isolation of weak or moribund animals.
(iii) Cageside observations should include, but not be limited
to, changes in:
(A) the skin and fur;
(B) eyes and mucous membranes;
(C) respiratory system;
(D) circulatory system;
(E) autonomic and central nervous system;
(F) somatomotor activity; and,
(G) behavior pattern.
(H) Particular attention should be directed to observation of tremors,
convulsions, diarrhea, lethargy, salivation, sleep, and coma.
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(iv) Individual weights of animals should be determined shortly
before the test material is administered, weekly thereafter, and at
death or at sacrifice.
(v) Food and water consumption should be determined weekly during
the study.
(vi) The time of death should be recorded as precisely as possible.
(5) Gross pathology. A gross necropsy of all animals should be per-
formed at the time of death or at sacrifice. All gross pathological
changes should be recorded.
(i) Techniques. The presence of the MPCA in tissues, organs,
and body fluids should be assessed by using sensitive, quantitative
techniques, in those animals that die during the study, and in all
animals that survive, at termination of the dosing period. Recovery
values and detection and sensitivity limits should be determined
and reported for each quantitative enumeration technique used.
(ii) Tissues, organs, and body fluids. The MPCA should be
enumerated from the kidney, brain, liver, lung, spleen, blood, and
representative lymph nodes. Other tissues, organs, and body fluids
may have to be examined as indicated by the nature of any toxic and
pathogenic effects observed.
(f) Data and reporting.
(1) Treatment of results. In addition to the information
provided recommended by 150A-4, the test report should include
the following information:
(i) the number of animals at the start of the test;
(ii) time of death of individual animals;
(iii) number of animals displaying other signs of toxicity and
pathogenicity;
(iv) description of toxic and pathogenic effects;
(v) definition for one unit of the MPCA used, and the units/test
animal in the dosing suspension;
(vi) body weights, food and water consumption;
(vii) necropsy findings;
(viii) pathology findings;
(ix) MPCA enumeration from tissues, organs, and body fluids,
and methods used, and sensitivities and limits of detection,
and;
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Subdivision M
(x) verification that each enumeration method is sufficiently
sensitive to serve as a useful quantitative assay for the MPCA.
in tissues, organs, and body fluids.
(2) Evaluation of results. An evaluation should include the
relationship, if any, between exposure to the test substance and
incidence and severity of all abnormalities, including:
(i) behavioral abnormalities;
(ii) clinical abnormalities
(iii) gross lesions;
(iv) body weight changes;
(v) food and water consumption;
(vi) mortality;
(vii) toxicity; and,
(viii) pathogenicity.
(g) Tier progression.
(1) If significant or persistent signs of pathogenicity are
observed in test animals, then consultation with the Agency is
required for determination of further testing requirements. Ihe
ability of the MPCA to overcome natural host barriers to infection
may be considered as a pathogenic trait, even when overt signs of
disease are not apparent.
(2) If toxicity effects are observed, in the absence of sig-
nificant pathogenic effects, then;
(i) the toxic component(s) of the dosing material are to be
identified, and to a practical extent, isolated, and;
(ii) an acute toxicity study ( 152A-20) is to be conducted
with the toxic components.
(3) If signs of infectivity, pathogenicity and toxicity are
not observed, then no further testing is required. However, the
registrant is required to provide an in-depth evaluation of possible
consequences of unusual persistence of the MPCA in host organisms.
(4) If significant infectivity is observed in the absence of
pathogenicity and toxicity, then a reproductive and fertility effects
study (152A-30) is required.
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Pesticide Assessment Guidelines
Tier III
152A-30 Reproductive and fertility effects of microbial pest
control agents rfMPCA)s1: Tier III.
(a) When required. Data from a one-generation reproduction
and fertility study are required by 40 CFR 158.740 to support the
registration of each end-use product that meets any of the following
criteria:
(1) Significant infectivity of the MPCA is observed in test
animals in the subchronic Tier II study (152A-21), and in which no
signs of toxicity or pathogenicity were observed.
(2) The MPCA is a virus which can persist or replicate in
mammalian cell culture lines ( 152A-19).
(3) The MPCA is not amenable to thorough taxonomic classifi-
cation, but is related to organisms known to be parasitic for mam-
malian cells.
(4) The MPCA preparation is not sufficiently well purified, but
it is indicated that the preparation may contain contaminants which
are parasitic for mammals.
(b) Purpose. This guideline for a one-generation reproduc-
tion/fertility study is designed to provide information on the
effects of a MPCA on fertility and on embryo/fetal development of
test animals. Effects of the MPCA on fertility and fetal development
to be evaluated include the number of non-pregnant females, numbers
that gave normal birth, number of resorptions, litter size, delayed
birth, embryolethality, and offspring body weight. Transmittal of
the MPCA from parent to offspring also is evaluated.
(c) Principle of the test method. The MPCA is administered to
male and female parents prior to their mating, and to maternal
parents during the resultant pregnancies.
(d) Substance to be tested. Testing shall be performed with
the technical grade of each active ingredient.
(e) Test procedures.
(1) Animal selection.
(i) Species and strain. The mouse or the rat are the preferred
species. Commonly used laboratory strains should be employed. If
another species is used, then justification/reasoning for the
alternate selection should be provided. All test animals should be
free of parasites or pathogens. Strains with low fecundity should
not be used.
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Subdivision M
(ii) Age. Test animals should be between 6 and 8 weeks old
prior to the first dosing.
(iii) Sex. (A) Both males and females are to be studied for an
adequate assessment of the MPCA on fertility.
(iv) Numbers. Each test and control group should contain at
least 20 males and a sufficient number of females to yield at least
20 pregnant females at or near term.
(2) Control groups.
(i) A concurrent untreated control group is required.
(ii) A separate vehicle control group is not required except
when the toxicity of the vehicle is unknown.
(iii) A control group dosed with inactivated MPCA is recommended.
(3) Dosing, (i) Dose level. One dose level of at least 108
units of the MPCA per test animal should be used. Justification/rea-
soning must be provided if a dose level of at least 108 units test
animal is not used. Quantification of the units of the MPCA should
be done concurrently with dosing.
(ii) Dose route. Administration of the MPCA usually will be by
the oral route. If persistence or infectivity of the MPCA in the
Tier I studies was observed only after some other route of dosing
(e.g. intravenous), then this route must be used.
(iii) Dose frequency. The frequency of dosing with at least
108 units/ test animal should be such that a significant level of
MPCA is maintaijTPd in the parents prior to and during the mating
period, and in the female parents during pregnancy.
(4) Observation period. Duration of observation should be from
the initial dosing with the MPCA to sacrifice of the offspring.
(5) Observation of animals.
(i) A careful clinical examination should be made on each test
animal at least once each day.
(ii) Additional observations should be made daily with appro-
priate actions taken to minimize loss of animals to the study,
e.g., necropsy of, and MPCA enumeration from those animals found
dead, and isolation of weak or moribund animals.
(iii) Cageside observations should include, but not be limited
to, changes in:
(A) the skin and fur;
(B) eyes and mucous membranes;
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pesticide Assessment Guidelines
(C) respiratory system;
(D) circulatory system;
(E) autonomic and central nervous system;
(F) somatomotor activity, and;
(G) behavior pattern.
(H) Particular attention should be directed to observations of
tremors, convulsions, diarrhea, lethargy, salivation, sleep, and
coma.
(iv) Individual weights of animals should be determined short-
ly before the test material is administered, weekly thereafter, and
at death or at sacrifice.
(v) The time of death should be recorded as precisely as possible.
(6) Mating procedure.
(i) Parental
(A) For each mating, each female should be placed with a
single, randomly selected male until pregnancy occurs or three weeks
have elapsed. Mixed matings with other males should be avoided.
(B) Those pairs that fail to mate successfully should be eval-
uated to determine the cause of the apparent infertility. This may
involve such procedures as additional opportunities to mate with
other sires or dams, examination of the reproductive organs, and
examination of the estrus or spermatogenic cycles.
(C) Each morning, the female should be examined for presence
of sperm or vaginal plugs. Day 0 of pregnancy is defined as the
day vaginal plugs or sperm are found.
(ii) Special housing. Near parturition, pregnant animals should
be caged separately in delivery or maternity cages and provided
with nesting materials. The cages and materials should be free
from contamination by the MPCA. Dosing should cease prior to iso-
lation of the pregnant females.
(7) Observation of pregnant females.
(i) Food consumption and prolonged parturition should be
recorded, in addition to the above clinical observations (5i-v).
(ii) The duration of gestation should be calculated from Day 0
of pregnancy.
(iii) Each litter should be examined as soon as possible after
delivery for the number of pups, stillbirths, live births, and the
presence of physical and behavioral abnormalities.
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Subdivision M
(iv) Litters should be weighed at birth or soon thereafter.
(8) MPCA enumeration in parents and offspring.
(i) Infectivity or persistence should be assessed by using
sensitive techniques to determine, as quantitatively as possible,
the presence of the MPCA in test animals.
(ii) Organs, tissues, and body fluids of each male parent
should be assayed at the time when it is confirmed that the female
of the mated pair is determined to be pregnant.
(iii) Organs, tissues, and body fluids of each female parent
should be assayed as soon as possible after birth of the litter.
(iv) Quantitative determinations of the MPCA in the pups from
each litter should be .made on postpartum day 1.
(f) Data and reporting. (1) Evaluation of study results.
(i) An evaluation of test results shall include a reporting of
any and all effects of the MPCA on the test animals, all observations
made, statistical analyses, MPCA quantification in the dosing pre-
parations and in the parents and offspring, evidence that a signifi-
cant level of the MPCA was maintained in the parents, dosing schedule,
and animal weights. This should include an evaluation of the rela-
tionship, or lack thereof, between carriage of the MPCA by parents
and abnormal effects on reproduction and fertility.
(2) Test report. In addition to the information recommended by
150A-4, the test report shall include the following information:
(i) fertility indices and length of gestation;
(ii) species and strain;
(iii) time of death during the study or whether .animals survived
to termination;
(iv) effects on reproduction and on offspring
(v) time of observation of each abnormal sign, including
pathogenicity, and its subsequent course;
(vi) body weight data for parents and offspring;
(vii) necropsy findings;
(viii) MPCA enumerations; and,
(ix) statistical treatment of results, where appropriate.
(g) Tier progression. Any further testing that is to be required
will be determined after consultation with the Agency.
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Pesticide Assessment Guidelines
152A-31. Qncogenicity study with micrbbial pest control agents
ff MPCA)sii Tier III
(a) When required.
(1) Data from onoogenicity testing may be required, as set
forth in 40 CFR 158.740, to support the registration of each end-
use product, or of each manufacturing-use product which legally may
be used to formulate such an end-use product, when the potential
for oncogenic effects in mammals is indicated by the presence of
certain viral components of the product.
(2) A potential for oncogenic effects exists when a component
of the MPCA formulation is a virus, and any of the following criteria
are met:
(i) the virus is an intended or an unintended ingredient in
the product and is known to be oncogenic for mammals, or is suffi-
ciently closely related to such viruses.
(ii) the virus is an intended or unintended ingredient in the
product and is demonstrated to transform mammalian cells in cell
culture tests ( 152A-16); and,
(iii) efforts at characterization of a virus component of the
product are insufficient to allow for the conclusion that the virus
is potentially oncogenic.
(b) The study is not intended for evaluating the oncogenic
potential of any product that may be due to chemical components,
whether or not they are produced by a MPCA.
(c) Test standards. Since test standards will be developed on
a case-by-case basis, consultation with the Agency is advised before
performing an oncogenicity study.
152A-32. Immunodeficiency studies with microbial pest control
agents [fMPCA)s]; Tier III
(a) When required.
(1) Data from immunodeficiency testing may be required to
support the registration of each end-use product or of each manu-
facturing-use product which may legally be used to formulate such
an end-use product, when the potential for causing a state of immu-
nodeficiency in mammals is indicated.
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Subdivision M
(2) A potential for induction of immunodeficiency in mammals
exists when a component of the MPCA is a virus, and the virus is,
or is related to, any virus that is known to interact with components
of mammalian immune systems and cause a state of immunodeficiency.
(b) Test standards. Since test protocols will be developed on a
case-by-case basis, consultation with the Agency is advised before
performing immunodeficiency studies.
152A—33. Primate infectivity/pathogenicity with microbial pest
control agents [ (MPCZO s]: Tier III
(a) When required. Data from primate testing may be required
to support the registration of each end-use product or of each
manufacturing-use product which legally may be used to formulate
such an end-use product when:
(1) the potential for causing infectivity, pathogenicity,
cncogenicity, or immunodeficiency is indicated by the presence of
certain intracellular parasites in the pesticidal product.
(2) a potential for adverse effects in primates exists when a
component of the MPCA formulation, during at least some stage in
its life cycle, can be an intracellular parasite of mammalian
cells, and any of the following criteria are met:
(i) A component of the MPCA formulation is a virus, and the
virus is able to cause cytopathic effects in mammalian host cell
lines in cell culture tests ( 152A-16) and, in addition, is demon-
strated to replicate in mammalian host cells.
(ii) A component of the MPCA formulation is a known parasite of
mammalian host cells.
(3) unresolved positive pathogenic effects from Tier I tests
might be specific to the test animal (s) used.
(4) the taxonomic properties of the MPCA indicate that human
pathogenicity might be of significant concern.
(b) Test standards. Since test protocols will be developed on a
case-by-case basis, consultation with the Agency is advised before
performing this study.
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pesticide Assessment Guidelines
Series 153A: RESIDUE ANALYSIS GUIDELINES FOR
MICROBIAL PEST CONTROL AGENTS
153A-1 Overview.
(a) Requirements. A petition for a tolerance or for
an exemption from the requirement of a tolerance must be
submitted as specified in 40 CFR 158.740 in connection
with each application for registration of a microbial pest
control agent (MPCA) where usage may result in residues in
or on food for humans or feed for domestic animals used for
human food. This petition mist contain data satisfying the
requirements of 40 CFR 158.740 which are detailed in this
section series (153) unless specifically exempted from the
requirements.
(b) Purpose. Residue chemistry data are designed to
provide the information necessary to determine the site,
nature, and magnitude of residues in or on food or feed.
This information includes plant metabolism data, residue
data, analytical methodology, and, when indicated, animal
metabolism data and animal feeding studies to determine the
carry over of residues into meat, milk, poultry, and eggs.
(c) Authority. Pesticides including MPCAs, intended
for use on food or feed crops, or where usage may reasonably
be expected to result (directly or indirectly) in residues
in food or feed, will not be registered unless a tolerance,
or an exemption from the requirement of a tolerance, has
been established by the Agency, as provided for under Sections
406, 408, or 409 of the Federal Food, Drug, and Cosmetic
Act ("FFDCA" 21 U.S.C. 346, 346a and 348). The procedural
regulations for filing petitions for a tolerance or an
exemption are included in 40 CFR 180.7.
(d). Approach. The use of a microbial pest control
agent on food, feed, or raw agricultural commodities requires
that a tolerance, or an exemption from the requirement for
a tolerance, be established by the Agency. In considering
exemptions from the requirement for tolerances, the Agency
recognizes that MPCAs do not necessarily pose the same
potential hazards as conventional chemical pesticides. In
fact, certain characteristics of many of these agents
suggest that they may pose relatively less hazard. These
characteristics are listed below:
(1) The efficacy of the agent often depends upon its
ability to replicate in the target pest, which is not likely
to remain on the crop after harvest.
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Subdivision M
(2) The living form of the agent in most instances
will usually not replicate in the absence of the specific
target pest (e.g., insect host).
(3) Certain environmental conditions such as sunlight,
rainfall, winds, humidity, and temperature often greatly
reduce the viability of the agent; therefore, the residues
of living organisms are apt to be small or relatively insig-
nificant shortly after application.
(4) Data supporting currently registered MPCAs indicate
that microbial pest control agents would not likely pose a
hazard to humans or other mammals.
(5) In many instances where and when a microorganism
is used as a microbial pest control agent, the microorganism
is already normally present in the environment and has
demonstrated no adverse effects.
(6) Residues of microorganisms used as microbial pest
control agents that are capable of replication on food or
feed—a very remote possibility—will possibly be rendered
nonviable or be removed by the usual processing of such
foods and feeds (i.e., washing, drying, heat sterilization,
and additions of sugar, salt, and other preservatives).
(e) Tier Progression. The Agency evaluates residue
data for microbial pest control agents used on food, feed,
or raw agricultural commodities only if toxic or other
harmful properties were observed in the maximum hazard
toxicology tests (Tier I) prescribed in 152A-10 through
-19 of this subdivision. If Tier I toxicology tests indicate
no toxic or other harmful properties, then no residue data
(with the general exception of a monitoring method) would
be indicated and thus a recommendation for an exemption
from the requirements of a tolerance can be made.
(f) Manor Issues. In many cases, a natural population
of microbial agent may be present at some background level
at the site where a microbial pest control agent is applied.
It may therefore be impossible to distinguish between natural
and introduced microbial populations and therefore be very
difficult to establish and enforce tolerances for naturally
occurring MPCAs. The Agency invites comment concerning the
testing methods for establishment and enforcement of tolerances
for naturally occurring MPCAs.
153A-2 [Reserved]
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Pesticide Assessment Guidelines
153A-3 General residue data requirements for microbial
pest control agents.
(a) When required. Residue data are required by 40 CFR
158.740 to be included in a petition for a tolerance or for an
exemption from the requirement of a tolerance, in connection with
each application for registration of a manufacturing-use product or
end-use product composed of or containing an MPCA, when the following
conditions are met:
(1) When the product is intended for use on food or feed
crops; or
(2) When use of the product is expected to result in residues
in or on food or feed; and
(3) When results of Tier I toxicology studies conducted in
accordance with 152A-10 through -19 of this subdivision indicate
that there may be significant human health concerns.
(4) Residue data will not be required and an exemption from
the requirement of a tolerance will be recommended for products
intended for use on food feed crops or for uses expected to result
in residues in or on food or feed, when the toxicology data developed
from Tier I testing, in accordance with 152A-10 through -19 of
this subdivision, indicate that there are no significant human
health concerns. The exception to this is that a monitoring method
will be required for each registered MPCA, even if exempt from tolerance.
(b) Procedures, standards, and reporting. In addition to the
provisions set forth in 150A-3 and -4 that are applicable, the
following guidance paragraphs are provided for conducting, developing,
and reporting the residue data that the Agency requires to support
a petition for a tolerance or for an exemption from the requirement
of a tolerance. In general, the guidance in Subdivision O (Series
170), particularly in terms of rationale and approach, is applicable
to MPCAs under this subdivision. Unless addressed below, all
paragraphs of Subdivision O as written are to be considered applicable
to this subdivision; the term "pesticide" is assumed to include
MPCAs. Discussion with appropriate Agency scientists may be helpful
before steps are taken to develop residue data of the nature outlined
in this series.
153A-4 Chemical Identity
In addition to the information detailed under 171-2 of
Subdivision O, the MPCA must be identified and characterized. If
the MPCA is a genetically altered microbe, then the nature and
source (if applicable) of the inserted or deleted genetic material
must be provided. The number of viable, infectious, or replicative
entities (propagules) per unit weight or volume of product must be
specified as well as the density of the product if it is a liquid.
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Subdivision M
153A-5 Directions for Use
In addition to the guidance under 171-3 of Subdivision 0,
the number of prqpagules per acre proposed for use is to be specified.
153A-6 Nature of the Residue in Plants
(a) When Tiequired. Plant metabolism studies are required to
determine the nature of the MPCA residues in plants whenever an
MPCA use is determined to be a food use. If a use is likely to
result in MPCA residues in or on food, then this is considered to
be a food use. In some cases, however, it may be possible to waive
the requirements for plant metabolism data if no potential MPCA
residues are expected to be of toxicological concern; such a waiver
is most likely to be granted for an indigenous microbe that does
not produce or code for a toxin. Attempts must be made to characterize
all MPCA residues in or on plants regardless of their route of
entry and/or deposition or whether they were produced via metabolic
or strictly physicochemical processes.
(1) Potential residues of concern may be, but are not
limited to, the following:
(i) all types of propagules of the parent (active ingredient)
MPCA such as vegetative cells, sexual and asexual spores, virions,
viroids;
(ii) mutants of the MPCA in question;
(iii) the genetic material itself;
(iv) any antigenic /allergenic, toxic, and/or mutagenic
substances associated with the MPCA product either as impurities in
the formulation or as components or metabolic products of the MPCA
itself produced during the manufacture or storage of the product or
at the site of application;
(v) or any other replicating entity which is the recipient of MPCA
genetic material (chromosomal or extrachromosomal) of potential
concern (such recipients may be viruses or cells of animals, plants,
or bacteria). The genetic stability (ease of genetic exchange or
transfer) is thus important in determining potential "residues" of
concern, especially in the castes of nonindigenous or genetically
altered MPCAs.
(b) Test procedures and reporting of data. The uptake,
metabolism, and translocation of the MPCA must be determined,
typically in a representative member of each crop group defined in
40 CER 180.34 (f) except the Herbs and spices group. If the metabolism
is similar in three unrelated crops, then additional crops normally
need not be included. In the case of demonstrated or potential
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Pesticide Assessment Guidelines
plant pathogens, however, more extensive testing may be required,
especially since replication of the MPCA may occur in nontarget
crop species; consultation with appropriate Agency scientists should
be sought if a petition involves a plant pathogen. If possible,
treated plants must be grown to normal crop maturity so that residues
may be characterized in or on the raw agricultural commodities
(RACs) derived from the crop in question (listed in Table II of
Subdivision 0).
Plants must be treated via the proposed route (seed treatment,
soil treatment, foliar plus soil treatment, etc.). Since intentionally
added inert ingredients could significantly influence MPCA deposition,
viability/stability, absorption, and even replication at the treatment
site, the test substance should be a typical end-use product (EP).
In some cases, radiolabeling of the MPCA may be useful but, generally,
other approaches must be employed to determine the total terminal
residue and, subsequently, which terminal residues are of toxicological
concern (refer to Series 152 of this subdivision for guidance).
The application rate should be the maximum proposed rate or exaggerated
rates, if necessary for residue identification.
MPCAs, being much more complex than conventional chemical
pesticides, create unusual analytical problems. For example, the
total terminal residue may consist of drastically different entities
(whole microbes, toxins, etc.) each requiring greatly differing
analytical procedures. Also, since all or most MPCAs are replicating
entities, the total terminal residue will frequently increase with
time but not as a function of continued residue absorption and/or
transformation of one residue to another as is typically the case
with conventional pesticides. In some cases, the viable MPCA per
se may not be of toxicological concern but a toxin produced by the
MPCA may be of concern. Note, however, that the MPCA itself will
be regulated even if it serves only as a source of a residue of
toxicological concern.
Some MPCAs may remain in the soil and/or on aerial plant parts
whereas others may gain entrance into plants via active or passive
routes. Once in the plant, the MPCA may be transported to other
plant parts, may replicate, and may cause disease; in other cases,
replication may occur only at the site of entrance. Replication
may occur only in arthropod or other hosts in or on plant tissues.
In some cases, the MPCA may be restricted to the soil or an isolated
plant part, perhaps not a PAC, but exert its influence throughout
the plant such as in the case of a translocatable toxin. MPCA
residues may fluctuate with the growth cycles. It is the responsibility
of the petitioner to demonstrate which, if any, plant parts bear
which residues of concern, i.e. to determine the distribution of
residues. These studies will demonstrate which are the major
residues to be sought in field residue studies, if required, and
also will provide an indication as to the efficiency and sensitivity
of the methods used.- Refer to 153A-8 for a discussion of some
analytical methods expected to be of use for MPCA determination.
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153A-7 Nature of the Residue in Animals.
(a) When required. Animal metabolism studies are required to
determine the nature and distribution of the MPCA residues in
animals if the following conditions are met:
(1) MPCA residues have been determined to be of toxicological
concern, i.e. Tier II or III toxicology testing must be performed
( 152A-20 through -32 in this subdivision): and
(2) Residues of concern will be present in or on feed items
(see Table II of Subdivision O); or
(3) Direct animal or animal premise treatments are proposed.
These types of studies may be required even if no residues of
toxicological concern occur in or on feed items because animal and
plant "metabolism" may be different.
Waivers of these data requirements are possible if the petitioner
is able to adequately demonstrate that no residues of toxicological
concern will occur in animal products used for food. Refer to
153-6 (a) (1) for examples of potential residues of concern.
(b) Test procedures and reporting of data. If one
or more of the conditions above are met, then animal metabolism
studies must be conducted utilizing ruminants and poultry. If
residues of toxicological concern occur in or on feed items, then
animals generally must be dosed orally for at least 3 days with the
MPCA terminal residues in feed items characterized to the extent
possible. Note that, theoretically, a single MPCA propagule in or
on a feed item could cause infection or allow replication in livestock;
obviously, this single viable unit could be a residue of toxicological
concern but would most likely not have been detected in or on the
feed item. Therefore, if any potential for animal pathogenicity
exists, animal metabolism studies will be required in all cases.
If orally or dermally administered direct animal treatments or
animal premise treatments are proposed, then animals/premises must
be treated according to the proposed use directions or at exaggerated
rates (if necessary for residue characterization) using a typical EP
product.
If no replication of the MPCA occurs in or on the animal, then at
least muscle, fat, kidney, and liver must be analyzed within 24
hours of dosage cessation. If MPCA replication occurs in the
animal, then several longer intervals may also be required and more
extensive tissue sampling will be necessary. Some MPCAs, notably
fungi, may require weeks or months to establish a detectable
infection. Eggs and milk, where applicable, must be sampled at
regular intervals, preferably daily. If may be possible to combine
the animal metabolism and animal magnitude studies (153A-10 of this
subdivision), if required. These studies will allow the determination
of the major residues for which analytical methodology must be
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developed and those which must be sought in animal feeding studies,
if required. The efficiency and sensitivity of the utilized methods
will also be determined from these animal metabolism studies.
153A-8 Residue Analytical Methods.
Analytical methods are required both for data collection to
support proposed tolerances and for the enforcement of such regula-
tions (40 CFR 180). Note that a monitoring method is required for
the determination of all MPCAs that are exempt from the requirements
of tolerance; the Agency must have this monitoring method available
in timps of need and cannot afford the potentially long method de-
velopment period in the event adverse effects are observed subsequent
to registration. The method(s) must not be subject to interference
due to substrate, reagents, or residues (cells, virions, toxin,
etc.) of related or unrelated microbial agents whether naturally
occurring and unregistered or whether an MPCA. A confirmatory
procedure is also required for each residue of concern for both
data collection and tolerance enforcement.
Each method must be fully described or a reprint must be provided
as well as any necessary modifications. Each method must be
validated by submitting recovery data and analyses of untreated
control samples of representative plant and animal commcdities.
The estimated sensitivity/detection limit must be provided for each
tested commodity. Attempts must be made to determine if residues
in or on treated commodities (aged or weathered) are extracted with
the same efficiency as those from spiked samples used for recovery
experiments.
Widely differing analytical methods may be required to identify
and quantify all residues of toxicological concern derived from a
given MPCA. If a biologically active microbial product is of
concern, then the more conventional analytical procedures such as
gas dhromatography, mass spectrometry, or high-pressure liquid
cshromatography are typically used. If the MPCA per se, a mutant,
or a viable recipient of MPCA genetic material is a "residue" of
toxicological concern, then various immunological methods (such as
enzyme-linked immunosorbent assay, dot-imntunoassay) or molecular
probe methods (such as dot hybridization, Southern hybridization
procedure, or restriction endonuclease mapping) may be used for
identification and/or quantification. Since the above procedures
do not necessarily determine viable MPCAs, culturing of
tissues (maceration followed by dilution plating) or infectivity
assays will frequently be necessary. Culturing or bioassays are
also important as means of detecting MPCAs at levels below the
detection limits of the above methods and, theoretically, as few
as one viable microbe can be detected using enrichment techniques,
if necessary. In some cases, microscopy may be useful.
It may be possible to purify some viral MPCAs to the point of
crystallization whereas other MPCAs may not be isolatable from host
cells or host membranes in a viable form. Some MPCA residues may
be bound (actively or passively) to cellular structures/components
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Subdivision M
and their release must be attempted using procedures such as
sonication, use of detergents, or hydrolytic steps (enzymatic,
acid, or alkaline). Care must be taken to determine background
levels of cross-reacting MPCAs since antigenic similarities
and/or nucleic acid homology may exist in indigenous microbes to a
greater or lesser extent. In some cases, care must also be taken
to detect different viable forms of the MPCA in question (such as
spores vs. vegetative cells, encapsulated vs. nonencapsulated, or
"yeast" vs. mycelial forms) since antigenic determinants may be
different or may be masked. In the case of genetically altered
MPCAs, the methods must be specific enough to determine the MPCA in
the presence of the parent, unmodified, Indigenous strain of the
same microbe which, generally, will differ only in a relatively
small portion of nucleic acid (chromosomal or extrachromosomal).
The regulatory method(s) must be relatively simple, rapid,
specific, and sensitive and should not require blank samples,
exotic equipment or reagents, or use of internal or procedural
standards. If the Agency finds the regulatory method (s) adequate,
it (they) will be published or referenced in the FDA Pesticide
Analytical Manual after an exemption from tolerance or a permanent
tolerance has been established.
153A-9 Storage Stability Data.
Storage stability data, as described in 171-4 (c) (1) (ii) of
Subdivision O, must be submitted. Studies involve spiking
representative plant and animal samples with each "residue" of
concern, storing under the same conditions (usually subfreezing
temperatures) as the treated samples, and analyzing at the end of
the treated sample storage period. It is imperative that samples
be stored at temperatures sufficiently low enough to prevent
replication of the MPCA or production of biochemicals of concern.
Note that freezing may be injurious to some MPCAs. If this is the
case, very short harvest-to-analysis intervals must be used or
other storage conditions must be devised.
153A-10 Magnitude of the Residue in Plants.
MPCA residues of toxicological concern must be determined
in or on PACs of all crops on which use is proposed or, if preferred,
RACs of each representative crop belonging to a given crop group under
40 CFR 180.34(f) to allow the establishment of a crop group tolerance.
If residues are detectable in or on the RAC or if concentration in
processed products is possible, then processing studies will also
be required. In general, the procedures described under 171-4 (c)
of Subdivision O should be used for guidance making certain that a
representative of each major formulation class is tested according
to the proposed use pattern. Table II of Subdivision 0 lists the
RACs and processed products of each agricultural crop. Note that
"pesticide" and "chemical" in Subdivision O are assumed to in-
clude MPCAs for purposes of this subdivision. Residue increases
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and/or decline in or on crops will frequently be a function of
growth or replication cycles of the MPCA which, in turn, is a function
of the population dynamics of potentially numerous other organisms
such as the host, the plant site (if different from the host),
other parasites/pathogens of either the host or the MPCA itself, or
competing occupants of the same or a similar habitate. It is ex-
pected that environmental conditions will play an even greater role
in the magnitude of MPCA residues in or on plants than in the case
of conventional chemical pesticides. Therefore, adequate geographic
representation of test sites is imperative; refer to USEA's Agricul-
tural Statistics (GPO), published annually, for the most recent
state production figures for various crops. Note that Subdivision
0 [ 171-4 (c)] states that a processing study utilizing radiolabeled
material may be required if processing of the FAC could result in
alteration of the residue; in the case of an MPCA, radiolabeling
will rarely be either possible or of utility and, therefore, other
approaches must be used.
In addition to the above information and the residue data
itself, the following materials and procedural details must be
provided: location of test, application rate (weight or volume of
product/A and number of viable microbes/A, etc.), formulation used,
part of crop analyzed, number of samples, sampling procedure, plant-
ing tiine (date), application date(s), harvest/sampling date(s),
application method, stage of crop at application and harvest, ana-
lytical method used, untreated control data, recovery data, and
sample storage time and conditions.
153A-11 Magnitude of the Residue in Animals.
Generally, the guidance presented in 171-4 (c) (3) of
Subdivision O is applicable to MPCAs under this subdivision noting
that, again, "pesticide" is assumed to include MPCAs. If residues
of toxicological concern occur in animal tissues, milk, or eggs
following oral dosing in the animal metabolism studies, then feeding
studies are required reflecting Ix, 3x, and lOx the maximum expected
dietary intake of MPCA residues occurring in or on feed items.
Feed items derived from each RAC are listed in Table II of Subdivision
O. Animals must generally be dosed for 28 days and slaughtered
within 24 hours of the final dose. If pathogenicity/ toxicity, or
very slow growth and/or disease development is a problem, then
appropriately shorter or longer feeding periods or perhaps even
longer preslaughter intervals may be used, preferably in consultation
with Agency scientists. The key issue is timing, i.e. the maximum
residue/MPCA concentrations in tissues, milk, and eggs must be
determined in order to allow the tolerances and, perhaps, preslaughter
intervals, to be established. Similarily, if residues occur in
animals following a direct animal treatment (if proposed) as
determined in an animal metabolism study, then studies utilizing
typical EPs must be conducted according to the proposed use
directions; such treatments could be feed-through, dermal, or
otherwise. If two or more routes of exposure are possible, then a
single study combining both routes are acceptable if the health of
the animals is not affected. Note that the petitioner may find it
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Subdivision M
advantageous to combine the animal metabolism studies (153A-7 of
this subdivision) and the feeding/direct animal treatment studies.
The dosage rate must be clearly stated. In the case of a feed-
ing study, ppn of nonviable residues and enumeration of viable
residues (colony- or plaque-forming units or infectivity units/unit
weight of feed or per unit weight per day) as well as the weights
of animals must be provided. In the case of direct-animal treatments
(oral, dermal, or otherwise), the weight or volume of product (typical
EP) and the number of viable MPCAs per unit weight, surface area,
etc. of animal, must be provided. Generally, ruminant and poultry
studies are required if residues of toxicological concern occur in
feeds or if direct animal treatments are proposed. Swine studies
may also be required if there is any reason to expect higher residues
and/or more rapid MPCA replication in swine than in poultry or
ruminants. If agricultural premise treatments are proposed, then
studies may be required to demonstrate the magnitude of the residue
in animals following these uses. All tissues used as food must be
sampled; eggs and milk must be sampled twice daily. Sample storage
time and conditions must be provided as well as the identity of the
analytical methods used, recovery data, and control animal data.
Also, the formulation used and number of samples must be presented.
153A-12 Potable Water. Fish, and Irrigated Crop Studies.
The intent and guidance provided under 171-4 (c) (4) of
Subdivision O are generally applicable to MPCAs proposed or registered
for use in or near aquatic sites. Note that it is possible that
replication of the MPCA may occur in irrigation water, natural
waterways, fish (including crustaceans), or irrigated crops.
Therefore, rather than dissipating or becoming diluted, MPCA residues
of toxicological concern may actually increase in the new environment.
New metabolites of concern may actually form. Note that a typical
EP should be used rather than the 14C-labelled material described
in 171-4 (c) (4) for the fish metabolism study, if required.
153A-13 Food Handling Establishment Studies.
Refer to 171-4 (c) (5) of Subdivision O for guidance useful
in developing studies to support use of MPCA products in food
handling establishments.
153A-14 Practical Methods for Removing Residues That Exceed Any
Proposed Tolerance (Section E of a Petition).
Refer to 171-5 of Subdivision O for guidance.
153A-15 Proposed Tolerances (Section F of a Petition).
Refer to 171-6 of Subdivision O for guidance.
153A-16 Reasonable Grounds in Support of the Petition
(Section G of a Petition).
Refer to 171-7 of Subdivision 0 for guidance.
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pesticide Assessment Guidelines
153A-17 Exemptions from the Requirement of a Tolerance.
Refer to 171-8 of Subdivision O for guidance.
153A-18 Tolerances for Foreign Uses.
Refer to 171-9 of Subdivision O for guidance.
153A-19 Rotational Crop Tolerances.
Refer to 171-10 of Subdivision O for guidance. If viable
MPCAs remain in soil for 18 months or more or if the MPCA in question
replicates in one or more rotational crops or their associated
flora or fauna, then residue data must be collected as described
under 153A-10 and 171-10 of these guidelines.
153A-20 Tobacco Uses.
Refer to 171-11 of Subdivision O for guidance.
153A-21 Data Requirements for Food Use vs. Nonfood Use.
Generally speaking, data will be required to demonstrate
that a given vise on a food crop (seed treatment, grown only for
seed, fallow land, or nonbearing crop) is not a food use. Since
MPCAs are replicating entities, there are very few uses, if any,
which could not ultimately be food vises. Refer to 171-12 of
Subdivision O for guidance.
153A-22 Submittal of Analytical Reference Standards.
Refer to 171-13 of Subdivision 0 for guidance.
153A-23 Special Considerations for Temporary Tolerance Petitions.
Refer to 171-14 of Subdivision O for guidance concerning
residue chemistry data requirements needed to establish a temporary
tolerance in conjunction with an Experimental Use Permit (EUP).
153A-24 Presentation of Residue Data.
Refer to 171-15 of Subdivision O for guidance.
153A-25 Translation of Data.
Refer to 171-16 of Subdivision 0 for guidance.
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Subdivision M
Series 154A: NONTARGET ORGANISM HAZARD GUIDELINES
154A-1 Overview
The purpose of nontarget organism testing is to develop data
necessary to assess potential hazard of microbial pest control
agents (MPCAs) to terrestrial wildlife, aquatic animals, plants,
and beneficial insects.
(a) Approach. The Agency has concluded that at least some
test data on terrestrial and aquatic organisms should visually be
evaluated, regardless of the pesticide's site of outdoor application
and apparent potential for exposure. These data would be necessary
for the following reasons:
When a microorganism is applied as a pesticide, great numbers
are placed in the environment apart from its host, at a discrete
point in time (day of application), and spread over living and
nonliving components of the target site. Often, there will be spread
to adjacent areas, due to drift. Hence, in terms of numbers of
nontarget organisms exposed, number of different species exposed,
and the degree of exposure (number of microorganisms per nontarget
organism), exposure may be greater than under natural conditions.
In addition, data on toxic or pathogenic effects are essential for
hazard assessment purposes when terrestrial or aquatic organisms
are likely to be exposed to a MPCA, especially when no fate data
will be required by the Agency in the first tier of testing.
Pathogenicity and toxicity appear to be the major effects of
concern regarding exposure of terrestrial and aquatic organisms to
microbial pesticides. Therefore, the Agency has developed guidelines
that will allow hazard assessment of pathogenicity and toxicity
problems to be made. The Agency desires a high level of confidence
that no unreasonable adverse environmental effects will result from
actual use of MPCAs. Toward this end, the guidelines in Tier I
reflect a maximum hazard approach to testing. Negative results
from tests using this approach would provide a high degree of con-
fidence that no unreasonable adverse effects are likely to occur
from the actual use of MPCAs.
If unacceptable adverse effects are identified in Tier I
tests, then Tier II tests are performed to attempt to quantify
levels of the MPCA to which the susceptible nontarget species may
be exposed. Prior to registration of MPCAs, applicants would submit
Tier I data on nontarget organisms. However, environmental expres-
sion data (Tier II) may also be required on a case-by-case basis
for certain MPCAs which are determined to present unique concerns.
In addition, on a case-by-case basis, definitive Tier II data showing
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pesticide Assessment Guidelines
that the MPCA will not survive or persist in the environment to
which it is applied, can be submitted as support for a request for
waiver (40 CFR 158.45) of some or all of Tier I testing requirements.
In some cases, a subchronic test may serve to better understand the
effects observed at the Tier I level and might alleviate the need
for Tier II testing.
If the MPCA is found to persist or survive in the environment at
significant levels as shown by Tier II tests, Tier III studies are
designed to show effects of chronic exposure to these levels on
fish and wildlife. If it is indicated that there may still be a
problem, Tier IV studies (simulated or actual field studies) may be
able to determine if there is a problem under actual use conditions.
(b) Manor issues.
(1) Maximum hazard dosage levels. Unlike environmental
levels of chemical pesticides, which generally decrease following
application, the environmental levels of MPCAs and any associated
toxins may, at least temporarily, increase when the product is
effective. Therefore, the maximum hazard dose for Tier I testing
will be based on some safety factor times the maximum amount of
active ingredient (MPCA or its toxin) expected to be available to
terrestrial and aquatic plants and animals in the environment. The
target hosts (e.g., insects) are likely to contain the highest
concentration of the microbial pest control agent that will be
available to nontarget terrestrial wildlife and aquatic animals
following a pesticide application.
Avian wildlife will be exposed, most commonly, through the
diet (via infected insects) or through the respiratory tract (via
spray drift or aerosolization). The maximum amount of MPCA a bird
in the wild may consume is difficult to determine, but as much a 1
x 109 is possible. Due to anatomical constraints, the Agency recog-
nizes that dosing at this level can not always be achieved. Thus,
the recommended daily oral or injected dose should be calculated as
follows:
Max. daily MPCA 5 ml/kg Weight of
dose (units) = Concentration X Body X Test
in TGAI Weight Bird (kg).
Therefore, for a product whose TGAI contains 1 x 109 units/ml
and using a 25 gram bobwhite quail, the maximum daily dose would be:
(1 x 109 units/ml) (5 ml/kg) (0.025 kg) = 1.25 x 108 units of MPCA
This dose should be administered over a 5 day period so that
the total dose the bird would receive orally, over a five-day period,
would be 6.25 x 108 units.
Maximum doses for the respiratory administration should be
calculated in a similar manner except that the dosing volume
should be reduced from 5 ml/kg to 0.2 ml/kg.
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Subdivision M
Maximum hazard aquatic exposures must, in some way, account
for the fact that fish and aquatic invertebrates, are less mobile
than terrestrial species and, therefore, less able to avoid the
pesticide. In addition, under conditions of nutrient influx or the
presence of alternate hosts or target pests in aquatic ecosystems,
aquatic organisms may be exposed to elevated numbers of microbial
pesticides. It is recommended, then, that the maximum hazard exposure
be in the range of 1 x 106 units of MPCA per ml of water or in a
concentration 1000 tin*"* the calculated concentration after direct
application to a 6-inch layer of water at label rates if the micro-
organism produces a toxin. Aquatic exposure will simultaneous expose
fish by the dietary route.
The Agency realizes that it would be very difficult to establish
specific I£5o, EDso/ or LD=0 values (e.g., U^Q = 1000 mg/kg)
and 95 percent confidence limits for most microbial pest control
agents whose mechanism of action is pathogenicity, because test
data are not likely to exhibit a log-probit dose-response relation-
ship that is typical of chemical pesticides. Therefore, data that
establishes an I£50, ED50, or 11)50 'l:nat is greater than the maximum
hazard dosage level (e.g., 11)50 >1000 mg/kg) would often be adequate
for the purposes of hazard assessment. In most cases, testing at
one maximum hazard dosage level is expected to be sufficient to
evaluate effects for these MPCAs. MPCAs that are toxin-producing
are more likely to produce a log-probit response. Therefore, in most
cases multiple groups would be necessary in order to quantify the
hazard of these organisms. If there are no effects at the maximum
hazard dose, lower doses will not be necessary.
(2) Maximum hazard routes of administration. Various routes
of administration (dosing) are provided for in these guidelines and
are chosen to reflect "natural" exposure routes. The Agency believes
that these routes; oral and respiratory for birds, aquatic and food
exposure for aquatic organisms, and the oral route for insects can
best define the hazard to nontarget organisms in the wild.
Parenteral dosing, such as intravenous and intraperitoneal
injection, would provide a high degree of confidence that a particular
microbial pesticide would not cause adverse effects, if negative.
Positive results, on the other hand, given the complex and undefined
(exogenous protein, metabolic byproducts, etc.) components of
microbial pesticide preparations and the environmentally unrealistic
nature of the route, would be difficult to translate to effects on
species in the environment. However, due to the high degree of
confidence an injection test gives, it is being suggested as an
alternate exposure route in the Avian Acute Oral, Toxicity/Patho-
genicity Test whenever the microbial dosing prepation is sufficiently
free from exogenous protein and other contaminating substances so
that the test will not be confounded.
(3) Aae of the test animals. The Agency considers that
sufficient immunological and physiological differences exist between
immature animals and mature animals to suggest that immature animals
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Pesticide Assessment Guidelines
are potentially more susceptible to infection and possibly to the
effects of any toxin produced by the MPCA. Therefore, the Agency
has developed age guidelines for the test animals in Tier I tests,
and recommends the use of immature animals in keeping with the
maximum hazard approach to testing.
(4) Methods for detecting the microbial pest control agents.
Unlike toxicity tests where mortality can usually be determined by
observation, infectivity tests often require sophisticated assessment
methods for detecting sublethal pathogenic effects. These methods
may include serological or nucleic acid technology.
(5) Detailed test protocols. No standard, widely accepted,
laboratory validated, test protocols are available at this time to
evaluate the safety of microbial pest control agents to terrestrial
and aquatic animals. The EPA Office of Research and Development is
in the process of developing and validating detailed testing pro-
tocols for the Office of Pestice Programs (OPP). In the meantime,
the draft and final protocols, as they became available, may be
obtained on request from the Environmental Fate and Effects Division
of OPP.
(6) Length of tests. The guidelines provide that the duration
of all Tier I tests be about 30 days long. This should permit time
for incubation, infection, and manifestation of effects in the test
organisms for most microbial pest control agents. Some test species,
notable nontarget insects, may be difficult to culture and the test
duration has been adjusted accordingly. Recommended test durations
are included for each testing guideline.
Various authors have proposed test duration times for toxicity
and pathogenicity tests ranging from 14 to 35 days (Ignoffo 1973;
Ignoffo et al. 1975; Summers 1975). The Agency realizes that the
test duration period may be unnecessarily long, or may not be suf-
ficiently long enough to detect effects such as viral diseases that
recur after prolonged intervals of latency, e.g., Herpes zoster
(Fenner et al. 1974). At the present time, however, the Agency is
not aware of an accurate method to predict whether a virus detected
in a test organism will manifest latent effects. The Agency invites
comments on the proposed test duration period and the probability
of encountering MPCAs with latent effects.
(7) Control groups. Appropriate control groups are addressed
in the recommended guidelines for each test.
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Subdivision M
154A-2 Terrestrial Wildlife.
(a) Approach. These guidelines call for two tests on birds
for all MPCAs: an avian acute oral toxicity and pathogenicity test
(154A-16) and an avian respiratory pathogenicity test (154A-17).
The avian acute oral toxicity and pathogenicity test would provide
data on any toxic effects to avian wildlife from exposure to the
microorganism or any toxin it may produce. This test would also
provide data on pathogenic effects following an acute exposure
either by the oral (or injection) route. The duration of the study
would be about 30 days to allow for an incubation period prior to
onset of symptoms.
The avian respiratory pathogenicity test would provide data on
the pathogenic effects of the MPCA on birds following exposure due
to drifts or aerosolation. The guidelines for the duration of the
test and gross necropsies are similar to the avian acute oral toxicity
and pathogenicity test.
In both the acute dose and inhalation tests, gross necropsy,
histopathological examination and culture and isolation should be
performed on exposure site tissues and other organs showing anatomical
or physiological abnormalities. In some cases, such as viruses,
there is a preference for certain cell or tissue types. In cases
where tissue preferences are known or suspected, those tissues
should be examined whether or not gross anatomical or physiological
changes are seen.
(b) Tier Progression.
(1) Tier I. If no toxic or pathogenic effects are observed
after exposing birds to the microbial pest control agent via two
different routes of administration (oral and respiratory) at the
maximum hazard dosage levels, then no further testing of birds
would be indicated. If toxic or pathogenic effects are observed at
the maximum hazard dosage levels, then Tier II, environmental ex-
pression tests (155A), would be indicated. In some cases, a sub-
chronic test may serve to better understand the effects observed at
the Tier I level and might alleviate the need for Tier II testing.
Data on wild mammal toxicity and pathogenicity (154A-18) are
required on a case-by-case basis when data indicate that there is
considerable variation in the sensitivity of different maiumalian
species to the effects of a MPCA or where wild mammals would be
heavily exposed to the MPCA under normal use. The toxicity and
pathogenicity data in section series 152A of this Subdivision for
evaluating hazard to humans and domestic animals are normally ade-
quate to indicate hazard to wild mammals. If no toxic or pathogenic
effects are observed in these tests, then no further testing of
wild mammals would follow. If any effects are observed in tests on
wild mammals, then Tier II, environmental expression testing (155A),
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would be indicated. In some cases, a subchronic test may serve to
better understand the effects observed at the Tier I level and
might alleviate the need for Tier II testing.
(2) Tier II. The data outlined in Tier II are described in the
environmental expression testing sections (155A) of these Guidelines.
If the expression characteristics preclude exposure of the MPCA to
nontarget birds and mammals, then no further testing of these animals
would be indicated. If Tier II tests indicate that birds and mammals
will be exposed to the MPCA, then testing at Tier III would follow.
(3) Tier III. The types of effects reported in the Tier I
tests would determine which Tier III test(s) would apply. If adverse
effects are reported in Tier I tests, and Tier H tests indicate
exposure, then Tier III testing would be required. If reproductive
or fertility effects, or oncogenicity are reported in tests in
152A30, and -31 for evaluating hazards to humans and domestic
animals, then a long-term avian pathogenicity and reproduction test
(154A-26) would apply. This test would provide data on pathogenic
effects of the MPCA on birds during a critical period in their
life—breeding and reproduction. It would also provide data on the
effects of the MPCA on avian reproduction. If no pathogenic or
reproductive effects are observed, the Agency would, at this time,
review all the data and determine if decisions regarding registration
can be made.
Pathogenic effects occurring at Tier III and beyond raise
serious questions
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Subdivision M
(1) In vivo testing. The guidelines outline in vivo testing
of birds and mammals. In vitro testing may be considered in the
future. Wolf (1975) has suggested a two-pronged testing approach
for safety testing of baculoviruses, using both in vivo and tissue
culture testing. He reported that there are established or permanent
cell lines for duck embryo fibroblasts, chicken embryo fibroblasts,
and representative mammalian cell lines from a bat, rabbit, mouse,
and deer. Ignoffo (1973) reported that at least 12 viruses, including
all major viral types, have been tested in vitro in either avian
egg embryo fibroblasts (chicken or turkey), fish, or mammalian cell
lines. Virus multiplication or cytopathic effects were reported
for one nuclear polyhedrosis virus in chicken embryo cells and
human amnion tissue, and for one noninclusion virus in chicken
embryo cells and mouse sarcoma tissue. In contrast, no effects
were observed in vivo when rabbits and mice were injected or fed
the latter virus. More recently, Brusca, et al., (1986) have shown
that Autoqraphica californica NPV can penetrate the nucleus of 3
poikilothermic vertebrate cell lines, although no productive
infection was demonstrated.
The Agency is not convinced at this time that the results of
in vitro tests can be used exclusively to determine potential
adverse effects to individual terrestrial animals (e.g., endangered
species) or populations of terrestrial animals in the environment.
(2) Test substance. Microorganisms used as pesticides could
be applied in any one of a combination of naturally existing forms.
It is preferable that the test organism be exposed to the most
infectious form whenever infectivity is the primary hazard of
concern. Similarly, when toxicity (e.g., a microbial toxin) is the
hazard of concern, the test organism should be exposed to a form of
the MPCA in which the toxin would be produced in the greatest amount
and most readily available. Unfortunately, there is no easy way to
determine which is the most infectious or toxic form of the micro-
organism to the test organisms. The route of administration may
also play an important role in determining which form should be
tested. For example, if the route of administration is intravenous,
then the active vegetative cells of a bacterium, or the infectious
hemolymph may be more appropriate than vegetative cells or polyhedryda,
respectively.
For these guidelines, testing the technical grade of the
active ingredient applies in all tests except the simulated and
actual field testing (154A-33), where the use of the formulated
product applies in order to simulate or reproduce actual field use.
The Agency realizes that in some cases the technical grade of the
active ingredient and the formulated product may be identical.
(3) Route of administration. These guidelines outline testing
by oral gavage or by injection and via the respiratory tract. It is
important to note that the administration of test material to 14- to
28-day old birds by oral gavage will likely require the use of
small needles or cannulae with ball-tipped ends in order to prevent
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injury to the birds. Ignoffo (1973) reported that the following
groups of terrestrial animals have been tested ill vivo for effects
caused by entomopathogens:
Group Routes of Administration
Mammals (primarily - Diet, oral, inhalation, sub-
laboratory populations) cutaneous, dermal application,
intradermal, intraperitoneal,
intravenous, intracerebral,
intranasal, intramuscular,
eye application.
Birds (chickens - Oral, diet, intraperitoneal
and laboratory populations (chickens).
that are phenotypically
similar to wild species)
Since the gut normally provides such a radically different environment
from that in the rest of the bird or mammal body, and since insecti-
vorous birds and mammals can be expected to ingest large quantities
of actively growing itdcroorganisms when they feed on diseased insects,
the Agency believes that the oral route would be appropriate. The
dietary route of administration was considered for Tier I tests,
but the Agency believes that it does not generally reflect the
maximum hazard test philosophy for Tier I tests. The diet, however,
is considered to be an appropriate route of administration for Tier
III and Tier IV tests (154A-26 and 154A-33).
The injection route may be used as an alternative route of
exposure for the oral toxicity/pathogenicity test, if the microbial
preparation does not contain excessive protein, metabolic byproducts,
etc. This route, although not environmentally realistic, provides
a maximum hazard challenge by bypassing the animals primary defense
mechanisms. If an injection route of exposure is used, a single dose
is acceptible in lieu of the multiple oral doses. Negative results
obtained by this dosing method indicate, with a high level of con-
fidence, that the MPCA under study will not produce adverse effects
to exposed avian species.
Inhalation, or rather intranasal or intratracheal instillation
has been chosen as the second exposure route because birds may
be exposed by this route during spraying operations or by the MPCA
made airborne through the effects of wind or animal movement during
feeding or other activities. In addition, the respiratory tract is
a major portal of disease acquisition in avian species.
The Agency is aware of the theoretical potential of microbio-
logical pesticides to disrupt the function of rumen bacteria. At
present, the Agency is seeking further information concerning the
possibility of such effects on wild mammals. If any such effects
were to be reported in safety tests on domestic ruminants, then the
Agency would solicit similar tests on wild ruminants.
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Subdivision M
The Agency recognizes that a combination of administrations in
one test (e.g., oral and intravenous or intraperitoneal injection)
may be possible. It would certainly be in keeping with the maximum
hazard testing philosophy and would reduce testing time and ex-
pense. However, combined exposures could unduly traumatize the
test animals so as to cause mortality, or in some other way cause
spurious results.
(4) Avian test species. These guidelines provide that young
bobwhite quail or mallard ducks be tested in Tier I tests. Birds
between 14 and 28 days of age at the beginning of the test period
should be used in the avian oral toxicity and pathogenicity test
and in the avian pathogenicity test. Within a given test, all
birds should be the same age (U.S. Environmental Protection
Agency 1978).
Summers et al. (1975) suggest testing two species of birds
including at least one insectivorous species. Wolf (1975) stresses
that test organisms should represent insectivorous and herbivorous
species. He suggests testing blackbirds, yellow-billed cuckoos,
representative members of the swallow family, and ducklings.
In Subdivision E of the Guidelines, the Agency suggests bob-
white quail, ringneck pheasants, and mallard ducks as acceptable
test species for avian acute toxicity tests of chemical pesticides.
The following facts influenced the Agency's proposal to test
bobwhite quail and mallard ducks in avian toxicity and pathogenicity
tests of MPCAs:
These species are ecologically significant and widely distributed
in the United States. They have proven to be good laboratory test
species and are appropriate for acute, subacute, and chronic testing.
laboratory populations are comparable to wild species. There is a
large amount of baseline necropsy and histological data available.
It has not been determined if any avian species or group of avian
species is a better indicator of potential effects from microbial
pesticides than bobwhite quail or mallard ducks, and, finally,
testing species from the family Icteridae (e.g., blackbirds, grackles,
and cowbirds) may be ecologically significant and in line with the
maximum hazard philosophy but would present many practical problems
in rearing, reproduction, controls, and handling.
In support of testing immature birds in Tier I, the Agency
notes that insects are vital to immature birds during the first 2
or 3 weeks of life, and make up a much larger proportion of their
diet during this time than at other times in their life. Thus,
they are functionally insectivorous birds at this age. Also, for
the purposes of pathogenicity testing, the Agency feels that suffi-
cient immunological and physiological differences exist between
immature birds and adult birds to warrant considering the immature
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pesticide Assessment Guidelines
bird as potentially more susceptible to infective challenge and so
proposes their use in the maximum hazard testing approach.
(5) Selection of dose levels. For Tier I tests, the Agency
suggests that a maximum hazard dosage be administered. For all
testing, the maximum dose should be no less than the maximum hazard
dose as defined in the testing guidelines (154A-l(h)l and 154A-16
to 154A-24) . If the MPCA produces significant toxic or pathogenic
effects at the maximum hazard dose level, then testing at lower
doses would be indicated. Sufficient doses and test organisms
would be required to determine an U^Q value, if possible.
(6) Protocols. Interim protocols for some ecological effects
testing have been developed by EPA's Office of Research and Development.
Although these protocols have not been validated, they are available
on request from EPA in order to provide guidance for applicants and
testing laboratories in developing protocols for testing microbial
pesticides on nontarget organisms.
T literature Cited
(1) Brusca, J. ; Summers, M. ; Couch, J. ; Courtney, L. (1986)
Autographa californica nuclear polyhedrosis virus efficiently enters
but does not replicate on poikilothermic vertebrate cells. Intervirology
26: 207-222.
(2) Friend, M; Trainer, D.O. (1974a) Experimental DDT-
Duck hepatitis virus interaction studies. J. Wildl. Manage.
38 (4): 887-895.
(3) Friend, M; Trainer, D.O. (1974b) Experimental
Dieldrin-Duck hepatitis virus interaction studies. J. Wildl. Manage.
38 (4): 896-902.
(4) Fenner, F. ; McAuslon, B.R. ; Mims, C.A. ; Sanbrook, J. ;
White, D.O. (1974) The Biology of Animal Viruses. Second Edition.
Academic Press, NY.
(5) Ignoffo, C.M. , Garcia, C. ; Kapp, R.W. ; Coate, W.B.
(1975) An Evaluation of the Risks to Mammals of the Use of an
Entomopathogenic Fungus, Nomuraea rilevi. as a Microbial Insecti-
cide. Pages 354-359, In Baculoviruses for Insect Pest Control.
Safety Considerations. Selected papers from EPA-^USDA Working
Symposium American Society of Microbiology. Washington, D.C.
(6) Ignoffo, C.M. (1973) Effects of entomopathogens on
vertebrates. Annals N.Y. Acad. of Sci. 217:141-164.
(7) McLeese, D.W.; Zitko, V.; Peterson, M. (1979)
Structure-lethality relationships for phenols, anilines and other
aromatic compounds in shrimp and clams. Chemosphere 2:53-57.
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Subdivision M
(8) Nordlund, D.A.; Lewis, W. J. (1976) Terminology of
chemical releasing stimuli in intraspecific and interspecific
interactions. Qiem. Ecol. 2(2):211-220.
(9) U.S. Environmental Protection Agency. (1978) Registration
of Pesticides in the United States: Proposed Guidelines, Subdivision
E Hazard Evaluation: Wildlife and Aquatic Organism Federal Register
43(132) '.29724-29737.
(10) Slesin, L.; Sandier, R. (1978) Categorization of
chemicals under the Toxic Substances Control Act. Ecol. Law Quart.
7:359-396.
(11) Summers, M.; Engler, R.; Falcon, L.A.; Vail, P. (eds.)
(1975) Guidelines for Safety Testing of Baculoviruses. Pages 179-
184 In Baculoviruses for Insect Pest Control: Safety Considerations.
Selected Papers from EPA-USDA Working Symposium, American Society
of Microbiology. Washington, D.C.
(12) Wolf, K. (1975) Evaluation of the Exposure of Fish and
Wildlife to Nuclear Polyhedrosis and Granulosis Viruses. Pages 109-
111 In Baculoviruses for Insect Pest Control: Safety Considerations.
Selected Papers from EPA-USDA Working Symposium, American Society
of Microbiology. Washington, D.C.
(13) Weiss, B; Laties, V.G. (1979) Assays for Behavioral
Toxicity: A Strategy for the Environmental Protection Agency. Pages
213-215 In Test Methods for Definition of Effects of Toxic Substances
on Behavior and Neuromotor Function, Neuro-Behavioral Toxicology,
Vol. 1. Suppl. 1. ANKHO International Inc. Report No. EPA 560/11-
79-010.
154A-3 Aquatic Animals.
(a) Approach. The Agency has considered several criteria that
could be used to determine the extent of testing for effects on
aquatic animals in Tier I. These are: the site of application and
resulting potential for aquatic exposure; the natural geographic
distribution of the microorganism; the natural population level of
the microorganism compared with population levels likely after
application; and, the ability of the MPCA to survive and replicate
after application.
While all of these criteria are important, the Agency has
chosen site of application and its resulting potential for aquatic
exposure as the key criterion for establishing the extent of initial
effects testing for MPCAs. The rationale for selecting this single
criterion is that it directly addresses the most critical issue
regarding potential hazard: likelihood of exposure. Furthermore,
the other criteria would be implicitly considered in connection
with the criterion for site of application.
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Pesticide Assessment Guidelines
The Agency recognizes that considerable judgment will be
required to properly employ site of application as a criterion.
While many uses obviously entail direct application to water (e.g.,
mosquito control and aquatic weed control), the Agency also intends
that less obvious or borderline uses will be considered aquatic
uses. Some examples that fall into the latter category are ap-
plications to forests, drainage ditches, riverbanks, and partially
aquatic crops such as rice. Widespread applications to major crops
such as cotton, soybeans, and corn could also warrant expanded
testing if these crops are grown near bodies of water. To the
extent possible, the Agency will rely on its experience with the
classical chemical pesticides in distinguishing between terrestrial
and aquatic use patterns in borderline situations.
(b) Tier Progression.
(1) Tier I. For MPCAs applied in terrestrial use patterns
(where direct aquatic exposure is not anticipated), one freshwater
fish (154A-19) and one freshwater aquatic invertebrate (154A-20)
should be tested to assess toxicity and pathogenicity. For MPCAs
applied directly to fresh, estuarine, or marine waters, one additional
fish species and one additional invertebrate species should be
tested in Tier I. These tests should be conducted as 30-day static
renewal bioassays using one or a combination of methods to administer
the pesticide (e.g., aqueous or dietary) These tests should be
designed to simultaneously assess both toxicity and pathogenicity
as well as to detect and quantify the microbial agent in the test
animal. The concentration of MPCA in the water or food must be
monitored to insure that the test organisms are exposed to a suf-
ficient MPCA level throughout the test period.
No further testing would be indicated if: (1) results of the
Tier I tests indicate no toxic or pathogenic effects, and (2) host
range testing indicates that the MPCA has a narrow host range such
that crossover into nontarget aquatic invertebrates is unlikely.
If toxic or pathogenic effects are observed, then environmental
expression testing (Tier II) would generally be required. In some
cases, a subchronic test may serve to better understand the effects
observed at the Tier I level and might alleviate the need for Tier
H testing.
If host range testing implies crossover into nontarget aquatic
invertebrates, then additional aquatic invertebrate species (those
expected to be susceptible or likely to be exposed) would have to
be tested in Tier I, or as an alternative, Tier II testing would
have to be conducted. If tests on these additional species indicate
toxic or pathogenic effects, then testing at Tier II would be in-
dicated; if otherwise, then no further testing would be necessary.
(2) Tier II. The data for Tier II are described in environ-
mental expression testing sections (155A) of these guidelines. If
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Subdivision M
the environmental expression characteristics do not indicate exposure
of the MPCA to nontarget fish or aquatic invertebrates, then no
further testing of these animals would be indicated. If Tier II
tests indicate that fish and aquatic invertebrates will be exposed
to the MPCA, then testing at Tier III is indicated.
(3) Tier III. Whereas Tier I tests are designed to screen
MPCAs using a maximum hazard testing scheme, Tier III tests are
intended to more precisely evaluate and quantify the actual hazard
associated with the MPCA. The types of effects reported in Tier I
tests would help determine which Tier III test(s) would be required.
If only toxic effects are observed in Tier I tests, then the guide-
lines of 72-1 through -6 of Subdivision E would apply, and further
testing would proceed as in Subdivision E. If pathogenic effects
or both pathogenic and toxic effects are observed in Tier I, then
tests that could be indicated in Tier III are the following: (1)
Additional acute or subacute test(s) of fish or aquatic invertebrates
to evaluate the spectrum of susceptible nontarget species, or deter-
mine the susceptible route(s) of exposure, or determine the
dose-response relationship between the pesticidal agent and suscep-
tible nontarget organism; (2) Aquatic invertebrate range testing
(158A-27) and fish life cycle testing (154A-28); and/or (3) Aquatic
ecosystem disruption studies (154A-29);
If results of Tier III tests indicate no pathogenic effects,
then no further testing would be indicated. Conversely, if results
of Tier III tests, along with environmental fate data, indicate
toxic or pathogenic effects, then simulated or actual field testing
(Tier IV) may be warranted.
(4) Tier IV. Simulated or actual field testing (154A-34)
provides data on the pathogenic effects of the MPCA on fish and
other aquatic animals following field applications at actual use
rates. This test would apply when pathogenic effects are reported
in Tier III testing (154A-27 and -29) at levels equal to actual
or expected field exposure levels, and when the Agency is reasonably
confident that quarantine methods can confine the MPCA to the test
area and prevent contamination of adjacent areas. The specific
test requirements would be determined on a case-by-case basis after
consultation between the Agency and the registration applicant.
(c) Manor issues. This section identifies and discusses
issues regarding aquatic testing of MPCAs that may require further
research and development. Most of the issues stem from two problems:
(1) There are no standard widely accepted test protocols available
to evaluate the effects of MPCAs on nontarget aquatic animals; and
(2) There are some potential hazards associated with the use of
MPCAs that the Agency recognizes and for which practical methods of
evaluation are unavailable. The role of in vitro testing and Tier
IV testing is also discussed in this section.
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Pesticide Assessment Guidelines
(1) Issues associated with Tier I protocol. Useful Tier I
test protocols would simultaneously assess toxicity and pathogenic-
ity in aquatic animals. The maximum hazard test philosophy would
be exerted in terms of treatment level, method of pesticide adminis-
tration, and age of the test animal.
A Tier I test should be conducted as a static renewal bioassay.
The microorganisms should be administered: (1) as a suspension in
the water (aqueous exposure); (2) in the diet in the form of diseased
host insects or treated feed, or; (3) as a combination of both
routes of exposure.
If any test animals die during the test, the cause of death
(e.g., toxicity, pathogenicity) should be determined, if possible,
and reisolation of the microorganism from test organism tissues
should be attempted. This information would be used to determine
what further tests, if any, are warranted. Exposure and observation
should extend for at least 30 days for fish and 21 days for aquatic
invertebates. Individual test animals should be removed periodically,
if necessary, throughout the test period and at test termination
for examination to assess pathogenicity.
If a subletnal infection is observed in test animals prior to
test termination, it may be necessary to continue the observation
period in order to more adequately assess the significance of the
infection (e.g., will it be lethal?). Several published studies
address certain aspects of the above-described protocol: Committee
on Methods for Toxicity Tests with Aquatic Animals 1975; Ignoffo
et al. 1973; Van Essen and Anthony 1976; Wolf 1975; Lightner et al.
13 Couch et al. 1975; and Hetrick, et al. 1979.
The following paragraphs discuss, in more detail, some of the
Tier I aquatic organism tests.
(i) Test organisms. The guidelines provide that the species
tested be selected from the list of species reconimended by the
Committee on Methods for Toxicity Tests with Aquatic Organisms
(page 21) (1975), with the exception of goldfish (warmwater species;
bluegill sunfish, channel catfish, and fathead minnow: coldwater
species; rainbow trout, brook trout, echo salmon). These species
are desirable test organisms for several important reasons: (1)
They are used to evaluate chemical pesticides, and therefore EPA
has considerable background data on these species; (2) Standard
methods for the care and handling of these species are available;
and (3) The species are widely distributed, are generally available,
and have a variety of food habits and habitat requirements.
When possible, consideration should be given to testing species
representative of the geographic region or ecosystem where the MPCA
is to be applied. When applicable, species likely to prey upon or
scavenge the diseased target host animals should be tested.
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Subdivision M
Unless there are other overriding considerations, the rainbow
trout should be used as the freshwater fish test species. It is a
desirable test animal because: (1) it is partially insectivorous;
(2) no one species has been shown to be preferable in terms
of sensitivity to MPCAs; (3) there is considerable background data
on this species pertaining to its microbial diseases (Mann 1978) ;
and (4) standard tissue culture procedures are available for this
species (Wolf and Quimby 1969 and 1973).
Use of young fish (3 to 6 months old) is preferable since they
would be more likely to display a lethal pathogenic effect, whereas
older fish may become carriers.
Due to the broad phylogenetic spectrum from which to choose,
it is difficult to select the most appropriate aquatic invertebrate.
Generally, a test organism that is phylogenetically closest to the
target host should be chosen. Such a test organism would be the
most likely to be susceptible to infection by the MPCA. Therefore,
when evaluating a MPCA whose target host is an insect, it would be
appropriate to choose an aquatic insect (e.g., caddisfly) as the
nontarget aquatic invertebrate test species.
Daphnia. a Cladoceran, has the advantage of having considerable
background data for comparative purposes. Pound (1977) exposed the
entomopathogen Mattesia to Daphnia and observed a bioconcentration
effect. This resulted from the filter feeding habits of Daphnia
and is a desirable feature in terms of assuring that the test
animal ingests the microorganism. Both Daphnia and certain other
aquatic insects have the advantage of a short life cycle or aquatic
phase, and both undergo periods of natural stress and potential
susceptibility to the microorganism as a consequence of molting.
(ii) Method of MPCA administration. Two methods of pesticide
administration should be considered: (1) Suspension in the test
water (aqueous exposure); and/or (2) Dietary, in the form of diseased
target host animals or incorporation of the MPCA into a standard
feed.
When possible, both routes should be used simultaneously in a
single test to ensure that the most appropriate route of exposure
has been tested and to insure a maximum challenge. Different patho-
gens may be capable of infection by different routes of exposure so
that no single route may adequately screen all microorganisms.
Each of the proposed routes has certain advantages and disadvantages.
Therefore, a multiple route of exposure would be extremely beneficial
and cost effective in screening MPCAs.
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Pesticide Assessment Guidelines
Addition of the microorganism directly to the test water is a
routine procedure. It simulates the type of natural exposure that
could occur immediately after application of a MPCA. It also
simulates the route of exposure by which many known pathogenic
agents infect fish and aquatic invertebrates. However, care must
be taken to assure that a high concentration of microorganisms be
maintainpd in the test system and that this high concentration does
not lower water quality to an unacceptable level. Therefore, the
static revewal method is recommended. Use of this method will
insure that high MPCA concentrations and acceptable water quality
can be maintained.
Dietary exposure also simulates certain natural conditions.
In fact, it is perhaps the most important means of infection for
the normal hosts of entomopathogenic agents (Surtees 1971).
Therefore, its use in evaluating effects on nontarget fish and
aquatic invertebrates is logical. This route offers a further
advantage: It increases the possibility of exposing the test
animals to a different life stage of the microorganism than may be
present in the formulated product if diseased target hosts (e.g.,
insects) are used as the feed.
Finally, oral intubation of fish is another possible route of
exposure, and is one that has been used to evaluate microorganism
effects in fish (Savan et al. 1979; Narayanan et al. 1977). This
route has the advantage of assuring that a known amount of test
material is ingested. This advantage, however, does not outweigh
the risk of injury or undue stress that could result from using
this method. Therefore, the oral intubation method though acceptable
is not recommended.
(iii) Test substance. The substance to be tested will
depend in part on the method of pesticide administration used in
the study. It is essential to test the most challenging form of
the microorganism (in terms of pathogenicity or toxicity). It is
equally important to test the form of the microorganism to which
nontarget aquatic animals are most likely to be exposed. These
objectives should be achievable through the use of multiple routes
of administration, provided it is known which form is most challenging
and which form is most likely to be encountered by the nontarget
animal. The technical grade of the active ingredient should be
used for all exposures. The formulated product should be tested if
it is to be applied directly to water.
(iv) Selection of treatment concentrations. Treatment concen-
trations must be related to the number of microorganisms to which
aquatic animals may be exposed under actual use conditions. And,
in keeping with the maximum hazard philosophy, treatment concentrations
must be relatively high. Consideration must be given to the level
of exposure resulting from direct application as well as exposure
resulting from consumption of diseased target host organisms (usually
insects). Exposure in terms of frequency and number of microorganisms
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Subdivision M
could be extremely high in the latter case.
The highest feasible concentrations should be used in all
exposures. At a miniraum, the concentration for aqueous exposure
1X106 units/ml water or 1000 times the theoretical concentration
present in 6 inches of water immediately after a direct application
of the MPCA, at label rates, to 6 inches of water, whichever is
greater and attainable. However, the use of such a high concentra-
tion may be limited by its adverse effect on water quality such as
oxygen depletion and production of metabolic wastes by the micro-
organisms. Therefore, treated water in the test vessels should be
renewed frequently enough to maintain water quality and microorganism
concentration.
(v) Test duration. Exposure and observation must be extended
to at least 30 days (unless test animals die) to allow time for any
potential infection, microorganism replication, or pathogenic or
toxic effects to manifest themselves. If a sublethal infection is
observed, then the test should be extended to evaluate the signifi-
cance of the infection. Similarly, if test animals begin to die
near the end of the 30-day period, the test should be continued to
determine the fate of the remaining test population.
The 30-day test duration was selected on the basis of past
research [Sevan et al. (1979); Pound (1977); Van Essen and Anthony
(1976); and tightner et al. (1973)]; and the recommendation of
Summers et al. (1975). Certain factors may dictate that this
period be modified. For example, if infection and death of target
hosts is normally not evident for many days (i.e., 20 to 30), it
would be logical to lengthen the period of exposure for the test
animals. Conversely, a shorter period of exposure may be warranted
in tests using animals with short life cycles (i.e., Daphnia or
mysid shrimp).
(vi) Observation and examination of test animals. Daily
observations are required to record mortalities and note any
behavioral, pathogenic, or toxic effects. Test organisms must be
examined for infection or any microorganism-related effects periodi-
cally throughout the study and at test termination. The most diffi-
cult aspect of this requirement is the verification of the presence
or absence of an infection. The general methods of assessment that
may be required to make this determination include histopathology,
sexology, and nucleic acid hybridization and reisolation and iden-
tification of the microorganism from organ tissue. These methods,
and the situations in which their use may be appropriate, were
presented in the general discussion of nontarget organism hazard
testing, 154A-1 (b) (4).
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pesticide Assessment Guidelines
Undeen and Maddox (1973) vised the following criteria in their
work with Nosema alqerae to distinguish between a true infection
and microorganisms observed in the test animal. In a true infection:
(1) Both vegetative forms and spores had to be present in the
test animal; and (2) The number of spores recovered had to exceed
the number injected by 100X. This type of approach may be useful
for other microorganisms.
(2) Issues associated with Tier III test protocols. The
aquatic invertebrate host embryo larvae, fish life cycle, and aquatic
ecosystem tests in Tier III (154A-27 through -29) are similar to
the protocols that are referenced for these types of tests in Sub-
division E of the Guidelines (77-4 through -7). Generally accepted
standard protocols for conducting these studies with MPCAs have not
been developed. In fact, few, if any, such tests have ever been
conducted with MPCAs. Therefore, at the outset, the Agency recognizes
that new and different test designs and test parameters may be more
appropriate than modified Subdivision E tests. Research and methods
development are in progress and need to be completed in this area
before the Agency can publish specific recommendations concerning
protocols and tier progression.
(3) The role of in vitro testing. The Agency recognizes that
there are in vitro tests available to assess the infectivity of
certain microorganisms, one of which is tissue culture for viruses.
Cell lines are established for several species of fish (Wolf 1975),
and such a test might be a useful means of assessing infectivity in
certain situations. However, the relationship between effects
demonstrated by in vitro tests and effects likely to occur under in
vivo situations is uncertain. For example, Ignoffo (1975) states
that "Tissue, completely nonsusceptible in the intact organism, may
support viral multiplication when explanted into a culture media."
Therefore, the results obtained from tissue culture tests could be
useless in accurately predicting environmental hazard. Another
potential drawback of tissue culture studies is that, often, no
host cell culture (e.g., insect cell culture) has been developed.
Therefore, such a study would have no positive control group and
the validity of a negative result would always be subject to some
doubt.
The Agency has concluded that, at the present time, in vitro
studies such as tissue culture cannot be substituted for the in
vivo studies provided in Tier I. At the same time, the Agency
recognizes the potential value of these studies for the following
purposes: (1) As a relatively inexpensive and rapid means to screen
for potential infectivity in a broad spectrum of species; and
(2) As a test to support or check the results of in vivo tests.
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Subdivision M
Therefore, a provision for cell culture studies is included in
Tier III of the testing scheme.
(4) Tier IV Testing. The Agency recognizes the possible
shortcomings in using simulated or actual field tests (Tier IV) as
the final test of the safety of a MPCA. If an agent has progressed
through the tier system and requires a field test, it must have
displayed significant adverse effects in some or all of the previous-
ly conducted laboratory tests. This fact might argue against the
use of a field test, since such a test could release potentially
hazardous microorganisms, with the potential to proliferate in the
environment and pose widespread environmental risk, unless adequate
quarantine measures could be taken. Therefore, before any Tier IV
field test is to be undertaken, the applicant should discuss its
plans with the Agency concerning potential hazards. If the Agency
determines that a Tier IV field test would pose an unacceptable
risk, then the MPCA would not likely be acceptable for registration.
On the other hand, the Agency also recognizes the potential
value of Tier IV simulated or actual field tests as a further check
on the safety of MPCAs that demonstrate no hazard in Tier I tests,
or that demonstrate a hazard that could be adequately controlled by
quarantine methods in the field. These tests could be conducted
concurrently with full-scale efficacy testing, and the Agency would
strongly encourage such tests. This would provide the opportunity
to evaluate pesticidal effects (both direct and indirect) on a much
broader spectrum of nontarget species, under more natural exposure
conditions, than is possible in Tier I testing.
(5) Assessment of other potential hazards: opportunistic
infections and latent viruses. Opportunistic infections in non-
target aquatic animals are recognized by the Agency to be a potential
hazard. A similar concern is noted for latent viruses. Research
indicates that aquatic animals may be rendered significantly more
susceptible to microbial infection, (e.g., by viruses and bacteria)
when stressed by such factors as Aroclor 1254 (Couch and Courtney
1977), copper (Hetrick et al. 1979), temperature, salinity, pesticides,
and other pollutants (Snieszko 1974; Schwartz 1974). This increased
susceptibility raises several important questions: (1) What is the
likelihood of an opportunistic infection (from a microbial pest
control agent) occurring in a nontarget aquatic animal? What is
the significance of the effect of opportunistic infections on in-
dividuals and populations? (2) Will the proposed Tier I test
adequately screen MPCAs for potential opportunistic effects? Or
could a MPCA be noninfective in a Tier I test, but infect stressed
nontarget animals? (3) Will a latent virus be detected by a Tier
I test and, if so, how can its significance be assessed?
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pesticide Assessment Guidelines
There is far too little background information and research on
MPCAs to suggest an answer to the first question. However, the
Agency believes that the potential for this type of problem should
not be ignored. The Agency is confident that sublethal infections
produced in Tier I tests can be detected if the proper methods of
detection are employed. However, the potential for an apparently
noninfective agent (in Tier I testing) to infect stressed aniinals
is unknown. At present/ the Agency is not aware of any practical,
generally accepted, routine screening test that could be used in
Tier I to determine the potential for such an occurrence. If a
sublethal infection is observed in Tier I, then further testing may
be warranted. A id.croorganism/stress interaction test is proposed
in Tier III as a means of assessing sublethal infections, but further
research is needed to develop the protocol for such a test. With
regard to latent viral infections, the Agency is not aware of a
standard method to evaluate the potential for a latent virus to
reactivate and cause adverse effects in aquatic animals. Further
research is required.
(6) Qnoocrenic effects. The Agency recognizes the potential
for oncogenic effects that are associated with viruses and mycotoxins.
The probability of oncogenicity in nontarget aquatic animals, as a
result of exposure to a viral pesticide, is unknown. At this time,
the Agency is unaware of any standard method that could be used to
screen for such an effect. Further research is required to develop
an appropriate test and determine when its use is justified.
Literature Cited
(1) Committee on Methods for Toxicity Tests with Aquatic
Organisms. (1975) Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates, and Amphibians. U.S. Environmental Protection
Agency. Ecol. Res. Series. EPA 660/3-75-009.
(2) Couch, J.A.; Summers, M.D.; Courtney, L. (1975)
Environmental Significance of Baculovirus infections in estuarine
and marine shrimp. Annals NY Acad. Sci. 196:528-536.
(3) Couch, J.A.; Courtney, L. (1977) Interaction of
chemical pollutants and virus in a crustacean: A novel bioassay
system. Annals NY Acad. Sci. 198:4977-504?.
(4) Hetrick, F.M.; Kiittel, M.D.; Fryer, S.L. (1979)
Increased susceptibility of rainbow trout to infectious hematopoietic
necrosis virus after exposure to copper. Appl. and Envir. Microb.
37(2):198-201.
(5) Ignoffo, C.M. (1975) Evaluation of in vivo specificity
of insect viruses. In Baculoviruses for Insect Pest Control:
Safety Considerations, M. Summers et al. (eds.) Am. Sec. Microbiol.
Washington, D.C.
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Subdivision M
(6) Lightner, D.V.; Proctor, R.R.; Sparks, A.K.; Adams, J.R.;
Heimpel, A.M. (1973) Testing Penaeid shrimp for susceptibility
to an insect nuclear polyhedrosis virus. Environ. Ent. 2 (4):611-
613.
(7) Macek, K.J.; Petrocelli, S.R.; Sleight III, B.H. (1979)
Considerations in assessing the potential for, and significance of
biomagnification of chemical residues in aquatic food chains. ASTM,
STP. Pages 251-268, In American Society for Testing
and Materials, Philadelphia.
(8) Mann, J.A. (1978) Diseases and parasites of fishes: An
annotated bibliography of books and symposia, 1904-1977. Fish
Disease Leaflet 53. USDI. Fish and Wildlife Service, Washington,
D.C.
(9) Narayan, K.; Govinarajan, R.; Jayaraj, S.; Raj, S.P.;
Kutty/ M.N. (1977) Nonsusceptibility of common carp, Cyrinus carpio
L. to nuclear polyhedrosis virus Baculovirus amsacta of groundnut
red hairy caterpillar. Madras Agric. J. 642(6):411-412.
(10) Pound, J.G. (1977) Safety and potential hazards of the
entomopathogen Mattesia trogodermae to nontarget species. Ph.D.
dissertation, University of Wisconsin.
(11) Savan, M.; Budd, J.; Reno, P.W.; Darley, S. (1979)
A study of two species of fish inoculated with spruce budworm
nuclear polyhedrosis virus. Wildl. Diseases 15:331-334.
(12) Schwartz, J.J. (1974) Prevalence of pathogenic pseunomonad
bacteria isolated from fish in a warmwater lake. Trans. Am. Fish.
Soc. 103:114-116.
(13) Snieszko, S.F. (1974) The effects of environmental
stress on outbreaks of infectious diseases of fishes. J. Fish.
Biol. 6:197-208.
(14) Summers, M.; Engler, R.; Falcon, L.A.; Vail, P. (eds.)
(1975) Baculoviruses for Insect Pest Control: Safety Considerations.
Selected papers from EPA-USDA Working Symposium. American Society
of Microbiology. Washington, D.C.
(15) Surtees, G. (1971) Epidemiology of microbial control on
insect pest populations. Intern. J. Env. Studies 2:195-201.
(16) Undeen, A.H.; Maddox, J.V. (1973) The infection of
nonmosguito hosts by injection with spores of the microsporidan
Nosema alqerae. J. Invert. Path. 22:258-265.
(17) Van Essen, F.W.; Anthony, D.W. Susceptibility of
nontarget organisms to Nosema algerae (Microsporida: Nosematidae),
a parasite of mosquitoes. J. Invert. Path. 28:77-85.
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pesticide Assessment Guidelines
(18) Wolf, K. (1975) Evaluation of the exposure of fish and
wildlife to nuclear polyhedrosis and granulosis viruses. In Baculo-
viruses for Insect Pest Control: Safety Considerations, M. Summers
et al. (eds.) American Society of Microbiology. Washington, D.C.
(19) Wolf, K.; Quimby, M.C. (1969) Fish Cell and Tissue
Culture. Pages 253-305 In Fish Physiology. Vol. 3. W.S. Moor and
D.J. Randall (eds.) Academic Press, New York.
(20) Wolf, K.; Quimby, M.C. (1973) Towards a practical
fail-safe system of managing poikilothermic vertebrate cell line
culture. In vitro 8:316-321.
154A-4 Nontarcret Plant Testing.
(a) Approach. The plant testing scheme proposed herein is
based on the tier testing scheme for testing other nontarget orga-
nisms. Tier I screening tests incorporates maximum hazard single
dosing using a route of exposure most likely to show any potential
plant toxicity or pathogenicity. The duration of the test should be
sufficient to allow for manifestation of a delayed pathogenic res-
ponse. Tier II testing examines population dynamics to quantify
persistence and survival of the MPCA in the environment. In some
cases, a subchronic test may serve to better understand the effects
observed at the Tier I level and might alleviate the need for Tier
II testing. Tier III testing is designed to record a dose response
and determine if there is a minimum infective dose for any adverse
effects identified in Tier I tests. Tier IV testing, if still
needed for risk assessment, will include both exposure and hazard
testing under simulated or actual field conditions.
(b) Discussion and major issues. Diseases of commercially im-
portant plants have been intensively studied for decades and many
plant pathogens have been identified and subsequently well charac-
terized. Some plant pathogens have a very narrow host range and
may attack only one species of plant, whereas other plant pathogens
may attack a wide range of plant species. Still other microorganisms
have never been identified in association with disease in plants.
A thorough taxonomic description of the MPCA should allow deter-
mination of its similarity to known plant pathogens. MPCAs that
are similar to plant pathogens with very narrow host ranges may
only need to be tested for adverse effects against plants similar
to known hosts. MPCAs that are similar to wide range plant pathogens
may need additional testing to identify the complete host range. A
knowledge of the mode of action may assist in determining the extent
of testing needed for potential plant pathogens. Finally, MPCAs
that do not resemble any known plant pathogen may require little,
if any, plant testing. Micrcbial herbicides are designed to be
toxic or pathogenic to their target plants. This class of MPCAs
will require close scrutiny to ensure that nontarget plants are not
unreasonably affected, whereas microbial insecticides generally
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Subdivision M
would not be expected to have phytopathogenic properties, and would
not require as much testing.
A second factor in determining the extent of plant testing is
the anticipated exposure of plants to the MPCA as determined by the
use pattern, dissemination, and the persistence /survival in the
environment. For example, MPCAs that will not be disseminated to,
or do not survive in, aquatic environments will not need testing in
aquatic plants. A related factor is whether the MPCA is to be used
within its area of natural occurrence. Where an MPCA is proposed
for use in an area where it does not naturally occur, additional
plant testing may be warranted.
Another factor in selecting species of plants to be tested is
that of susceptibility to plant diseases. Genetically diverse groups
of plants are generally less susceptible as a species to any given
plant pathogen since there is a greater chance that a variety of the
species will be resistant to the disease. The most important group
of genetically identical (monoculture) plants are the commercial
agricultural crops. These plants should be given priority in testing
for plant pathogenicity because of both their potential susceptibility
and their commercial importance.
154A-5 Nontarqet Insects.
(a) Terrestrial insects.
(1) Approach. Assessment of potential nontarget insect hazard
from uses of MPCAs is made difficult by a number of factors: (1)
Most MPCAs will be specifically selected and/or designed for their
ability to control pest insects. As such, nontarget insects represent
the organism group most at risk, being, in most cases, relatively
closely related to the target organism. (2) While there are few
nontarget insects that have been shown to be economically important
to humans, there are many nontarget insects which have an iinportant
role in ecological processes and may benefit humans indirectly. (3)
Unlike chemical pesticides, many microbials will exert their effect
through pathogenicity as well as toxicity. The acute, short dura-
tion, first tier tests, which should suffice for hazard evaluation
for some chemical pesticides, will not be appropriate for microbial
agents. Adequate assessment of pathogenicity will demand time to
evaluate the MPCA for infectivity and for its ability to reproduce
or develop in the test insect. (4) The host range is an important
factor in hazard evaluation for a MPCA. A problem here is that
extrapolation, even across species lines, is often not dependable.
For this reason, the Agency will provide for testing with represen-
tatives from a number of "beneficial insect" taxa. Information
from these tests will be used in conjunction with host range data
(developed during efficacy testing) to develop a clearer idea of
the overall insect host range.
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pesticide Assessment Guidelines
The Agency is aware that this first tier of testing may, in
scans cases, be more extensive than the baseline data requirements
in Subdivision L. However, there should be very few microbials
which require effects testing beyond the Tier I level.
In view of the factors cited above, the tier-testing scheme
for MPCAs is based on a fairly extensive first tier. The purpose
of the Tier I testing is to assess toxicity and pathogenicity of
the MPCA to the honey bee and to three species of predaceous and
parasitic insects. Selection of the predator/parasite species to
be tested should take into account such factors as the likelihood
of exposure to the MPCA, phylogenetic proximity of the test species
to target pest species, and similar relationships. A rationale for
selection should be developed by the registrant.
Interim protocols for some nontarget insect effects testing
have been developed by EPA's Office of Research and Development.
Although these protocols have not been completely validated, they
are available on request from EPA in order to provide guidance for
applicants and testing laboratories in developing protocols for
testing microbial pesticides on nontarget organisms.
(2) Tier Progression.
(i) Tier I. Under these guidelines, toxicity/pathogenicity
tests on the honey bee and insect predators and/or parasites are
indicated for all MPCAs. Selection of predator and parasite species
for testing is made by the registration applicant. Species selected
should be representative of groups which will be exposed under the
conditions of proposed use, and which have some important relation-
ship with the target pest. Rationale for selection is to be provided
by the registrant. The main purpose of the Tier I testing is to
determine presence of toxic or pathogenic effects on representatives
of a few major orders of beneficial insects. As noted above, the
representative test species selected, in addition to the honey bee,
should be of some importance in the ecosystem to be exposed to the
microbial control agent. Data derived from Tier I testing will be
used in conjunction with available information on use pattern, host
range (specificity), fate, and other similar factors, to assess
potential for adverse effects. If data indicate no potential for
adverse effects, no further testing would be indicated. Should the
results of Tier I testing indicate toxic and/or pathogenic effects,
then Tier II testing (environmental expression) would follow. In
some cases, a subchronic test may serve to better understand the
effects observed at the Tier I level and might alleviate the need
for Tier II testing.
(ii) Tier II. . The data for Tier II are described in environ-
mental expression testing (155A) of these guidelines. If expression
characteristics preclude exposure, no further testing would be
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Subdivision M
indicated. If data indicate that nontarget insects will be exposed
to the MPCA, then the registration applicant should consult with the
Agency regarding possible Tier III testing.
(iii) Tier III. For all MPCAs, Tier III consists of advanced
tests specifically responding to adverse effects identified in earlier
tier testing. Such tests may be simulated or actual field tests, but
further research is needed to develop the protocols for such testing.
In any case, Tier III testing would be preceded by consultation with
the Agency.
(b) Aquatic Insects. Tier I testing, as outlined in the "Aquatic
Animal Tier Testing Scheme for Microbial Pest Control Agents" (154A-
1) will include toxicity/pathogenicity testing with Daphnia. or a
species of aquatic insect, or both, depending on use pattern. Detec-
tion of pathogenicity/toxicity in Tier I testing will automatically
lead to expanded testing which, if the impacted site is fresh water,
will most likely involve testing with aquatic insects.
TIER I
154A-16 Avian oral pathogenicity/toxicity test; Tier I .
(a) When required. Data on the oral pathogenicity of a MPCA to
birds are required by 40 CFR 158. 740 (d) to support the registration
of each end-use formulated product (EP) intended for outdoor application
and each manufacturing-use product (MP) that legally may be used to
formulate such an end-use product. See 40 CFR 158.50 and 158. 740 (d)
to determine whether these data must be submitted.
(b) Test standards. Data must be derived from tests that
satisfy the general test standards in 150A-3 of this subdivision
and the following test standards:
(1) Test Substance. The actual form of the material to be
regarded as the test substance is described in Section 154A-2(c) (2)
of this document. In addition, any substances used to enhance
virulence or toxicity should be tested along with the test substance.
(2) Species. Testing shall be performed on two avian species,
one insectivorous and one herbivorous, (preferably bobwhite quail
and mallard duck) . The use of two species of birds with differing
diets is recommended in order to take into account possible differences
in gastrointestinal physiology. Other species may be used but a
justification must be supplied based on increased susceptibility to
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Pesticide Assessment Guidelines
the Microbial Pest Control Agent (MPCA) or ecological considerations
which preclude the use of recommended species.
(3) Age. Birds used in this test shall be from 14 to 24 days
old at the beginning of the test period. Within a given test, all
birds shall be as near the same age as possible.
(4) Controls.
(i) A negative, nondosed control group should be performed.
(ii) An infectivity control group should be performed and
should be treated with MPCA inactivated in such a way as to retain
the structural integrity of the cell.
(iii) A control group in which the birds are dosed with sterile
filtrate from production cultures should be performed concurrently
with the test groups.
(5) Number of birds per dosage level. Each treatment and
control group shall contain at least 10 birds. When only one treat-
ment group is tested, at least 30 birds shall be tested at that
level.
(6) Mayiittum hazard dosage level. The highest oral dosage
level tested is defined by the following formula:
Max. daily MPCA 5 ml/kg Weight of
dose (units) = Concentration X Body X Test
in TGAI Weight Bird (kg).
When using injection routes, substitute the following for
"5 ml/kg Body Weight": intravenous, "0.5 ml/kg Body Weight"; and
intraperitoneal, "2 ml/kg Body Weight".
For MPCAs that produce a toxin, fractions of this dose should
be calculated for lower doses. This dose is then administered
daily for 5 days. A justification shall be provided to support any
reduction in the highest dosage level.
(7) Treatment conditions.
(i) If the MPCA produces a toxin, then a sufficient number of
treatment doses must be tested to determine toxicity as described
in paragraph (b) (9).
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Subdivision M
(ii) If the MPCA does not produce a toxin, or no toxin has
been identified, then a single group of birds may be tested at the
maximum hazard dose. If deleterious effects, due either to toxicity
or pathogenicity, are observed, sequentially lower doses should be
tested as described in paragraph (b) (9).
(8) Dosing reaimen. Birds will receive oral doses daily for
five days.
(9) Determination of an ITbg or IDsp. Ihe study endpoint must
be chosen to reflect the activity of the~specif ic microorganism
under test, i.e., if an MPCA is expected to produce a toxin and has
little or no infectivity, then the appropriate endpoint would be
death of the test organism. If, however, the major mechanism is
pathogenicity, then a more appropriate endpoint would be overt
symptomatology.
Ihe test data should establish that the avian oral 11)50 /
defined as the dose required to kill 50 percent of the test organisms,
or IDsQ' defined as the dose necessary to cause overt symptomatology
in 50 percent of the test organisms, is greater than the maximum
hazard dosage level. If the IDc0 or 1050 is lower than the
maximum hazard dose, then a definitive U^Q or ID^Q with confidence
limits should be established.
(10) Deration of test. Control and treated groups should be
observed for at least 30 days after dosing initiation. If sympto-
matology or toxic signs are manifest at the thirtieth day, observation
should continue until recovery, mortality or unequivocal moribundity
is established.
(c) Reporting and evaluation of data. In addition to the
information specified in 150A-4 of this subdivision, the test
report shall contain the following information:
(1) Species;
(2) Age of the birds tested;
(3) Mean body weights for each test and control group at test
initiation and weekly thereafter;
(4) Diet analysis (especially antibiotics);
(5) Pen dimensions;
(6) Ambient temperature and humidity;
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pesticide Assessment Guidelines
(7) Riotqperiod and lighting;
(8) Toted feed consumption for each test and control group at
weekly intervals;
(9) Method of test material preparation, concentration of the
MPCA and total dose;
(10) Amount of test material, dosed per bird;
(11) Amount of vehicle dosed per bird, if a vehicle other
than water is used;
(12) Number of birds per group;
(13) ID50 or ID50 in appropriate units with 95% confidence
limits if obtained;
(14) Methods used for calculation of LD50 or ID50;
(15) Slope of the dose response line, if obtained;
(16) Time and date of mortalities;
(17) Any signs of intoxication, abnormal behavior, and
regurgitation (if any occurs);
(18) Reports of any pathogenic symptomatology or pathological
changes;
(19) Results of gross necropsies and histopathological findings
conducted on enough birds to characterize any gross lesions including
attempts, using appropriate techniques, to reisolate the MPCA from
examined tissues.
(d) Tier progression.
(1) If any pathogenic symptoms or toxic signs are observed
at any dose level in this study, testing at Tier U, environmental
expression testing ( 155A-15 through -23), is required as specified
in 40 CFR 158.740. In some cases, a subchronic test may serve to
better understand the effects observed at the Tier I level and
might alleviate the need for Tier II testing.
(2) If toxic or pathogenic effects are not observed in this
Tier I study, additional testing at higher tiers ordinarily is not
required. The agency may require additional testing, however, if
it determines that there is a potential risk to birds despite the
negative Tier I results.
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Subdivision M
(e) References. The following references are provided for
use in the development of acceptable test protocols for conducting
an avian oral pathogenicity/toxicity test with a microbial pest
control agent:
(1) Friend, M., and D.O. Trainer. 1971. Experimental duck
virus hepatitis in the mallard. Avian Disease 16(4): 692-699.
(2) Hartmann, G.C. and S.S. Wasti. 1980. Avian safety of
three species of entomogenous fungi. Camp. Physiol. Ecol. 5(4):
242-245.
(3) Ignoffo, C.M. 1973. Effects of entomopathogens on
vertebrates. Annals N.Y. Acad. Sci. 217:141-164.
(4) Lautenschlager, R.A., and J.D. Podgwaite. 1979. Passage
of nucleopolyhedrosis virus by avian and mammalian predators of the
gypsy moth, Lymantria dispar. Environ. Entomol. 8(2) :210-214.
(5) Narayanan, K., G. Santharam, S. Easwaramoorthy, and S.
Jayaraj. 1978. Lack of susceptibility of poultry birds to nuclear
polyhedrosis virus of groundnut red-hairy caterpillar, Amsacta
albistriga (W.). Indian J. Exoer. Biol. 16(12):1322-1324.
(6) Podgwaite, J.D., and R.R. Galipeau. 1978. Effects of
nucleopolyhedrosis virus on two avian predators of the gypsy moth.
U.S.D.A. For. Serv. Res. Note, NE - 251, 2 pp.
(7) Summers, M., R. Engler, L.A. Falcon, and P.Vail, eds.
1975. Guidance for Safety Testing of Baculoviruses, Pp. 179-184 in
Baculoviruses for Insect Pest Control. Safety Considerations.
American Society for Microbiology, Washington,
(8) Wolf, K. 1975. Evaluation of the exposure of fish and
wildlife to nuclear polyhedrosis and granulosis viruses. Pp. 109-111
in Baculoviruses for Insect Pest Control. Safety Considerations.
American Society for Microbiology, Washington, D.C.
154A-17 Avian respiratory pathoqenicity test; Tier I.
(a) When required. Data on the avian acute inhalation patho-
genicity of a MPCA are required by 40 CFR 158.740(d) to support
the registration of each end-use formulated product (EP) intended
for outdoor application and each manufacturinguse product (MP) that
legally may be used to formulate such an end-use product. See 40
CFR 158.50 and 158.740(d) to determine whether these data must be
submitted. The Agency recognizes that this test protocol has not
yet been evaluated in the laboratory. An inhalation exposure may
be acceptable in lieu of instillation and, for some microorganisms,
the Agency may accept a request for waiver of this test with appro-
priate justification. It would be advisable to contact the Agency
before performing the respiratory testing.
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pesticide Assessment Guidelines
(b) Test standards. Data sufficient to satisfy the requirement
in paragraph (a) of this section must be derived from tests which
satisfy the purposes of the general test standards in 150A-3 of
this subdivision, and all the following test standards:
(1) Test substance. The actual form of the material to be
regarded as the test substance is described in Section l54A-2(c)(2)
of this document. In addition, any substances used to enhance
virulence or toxicity should be tested along with the test substance.
(2) Species. Testing shall be performed on one avian species
(preferably bobwhite quail or mallard duck). Other species may be
used but a justification must be supplied based on increased suscep-
tibility to the MPCA or ecological considerations which preclude
the use of recommended species.
(3) Age. Birds used in this test should be from 14 to 28
days old at the beginning of the testing period. Within a given
test, all birds shall be as close to the same age as possible.
(4) Controls.
(i) A negative, non-dosed control group is required;
(ii) A concurrent control group is required and shall be
treated with the pure active ingredient that has been inactivated
in such a way as to preserve cellular integrity.
(iii) After dosing, two untreated contact control birds are
required and shall be placed in with the treatment group receiving
the maximum hazard dosage.
(5) Number of birds per dosage level. Each treatment and
control group shall contain at least 10 birds. When there is only
one treatment group at least 30 birds shall be tested at that treat-
ment level.
(6) Route of exposure. The test material should be administered
by intranasal or intratracheal instillation. Depending on the
physical properties of the agent being tested, an alternate route,
such as an aerosol, involving the respiratory tissues may be used.
However, use of the alternate route must be justified.
(7) Dosing regimen. Birds should receive doses daily for five
days.
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Subdivision M
(8) Maximum hazard dosage level. The maximum hazard dose is
defined by the following formula.
Max. daily MPCA 0.2 ml/kg Weight of
dose (units) = Concentration X Body X Test
in TGAI Weight Bird (kg).
Fbr MPCAs that produce a toxin, fractions of this dose should be cal-
culated for lower doses. A reason shall be provided to support any
reduction in the highest dosage level.
(9) Treatment concentrations.
(i) If the MPCA produces a toxin, then a sufficient number of
treatment concentrations shall be tested to determine toxicity as
described in paragraph (b) (10).
(ii) If the MPCA does not produce a toxin, or no toxin has
been identified, then a single group of birds may be tested at the
maximum hazard dose. If deleterious effects, due either to toxicity
or pathogenicity, are observed, sequentially lower doses should be
tested as described in paragraph (b) (10).
(10) Determination of an Tn5o or IPgn- The study end-
point must be defined to reflect the pathological activity of the
specific agent under test, i.e., if an MPCA is expected to produce
a toxin and has little or no infectivity, then the appropriate
endpoint would be death of the test organism. If, however, the
major mechanism is pathogenicity, then a more appropriate endpoint
would be overt symptomatology.
The test data must establish that the avian inhalation
LC50, defined as the dose required to kill 50 percent of the test
organisms, or IC50, defined as the dose necessary to produce overt
symptomatology in 50 percent of the test organisms, is greater than
the maximum hazard dosage level. If the I£^0 or IC50 ^ less
than the maximum hazard dose, then a sufficient number of treatment
levels should be tested in order to obtain a definitive I£50 or
IC50, if possible.
(11) Duration of test. Control and treated birds should be
observed for at least 30 days after dosing. If symptomatology is
manifest at the thirtieth day, observation should continue until
recovery, mortality or unequivocal moribundity is established.
(c) Reporting and evaluation of data. In addition to the
information specified in 150A-4 of this subdivision, the test
report shall contain the following information:
(1) Species;
(2) Age of the birds tested;
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Pesticide Assessment Guidelines
(3) Mean body weights for each test and control group at
test initiation and weekly thereafter;
(4) Diet analysis (especially antibiotics);
(5) Pen dimensions;
(6) Ambient temperature and humidity;
(7) Photoperiod and lighting;
(8) Total feed consumption for each test and control group at
weekly intervals;
(9) Method of test material preparation, concentration of the
MPCA and total dose;
(10) Identification of vehicle or carrier used to disperse
the agent and the average amount of vehicle administered to each
bird, if a vehicle other than water is used;
(11) Number of birds per treatment level;
(12) Number of controls used;
(13) Time and date of mortalities or symptom onset;
(14) IDsg or H>5o/ i*1 appropriate units, with 95% confi-
dence limits, if obtained;
(15) Results of gross necropsy and histopathological findings
conducted on all birds dying before termination of the test, on a
representative sample of those that survived, and on two contact
control birds. The necropsy report should include any evidence of
respiratory tract involvement and involvement at distant sites
including liver, kidney, spleen, cerebrospinal system, gastrointes-
tinal system. Blood samples should also be analyzed for abnormalities.
Results should also include any attempts, using appropriate techniques,
to reisolate the MPCA from examined tissues;
(16) A clinical assessment of any histopathological findings
and lesions noted;
(17) Assessment of the clinical significance of MPCA tissue
isolations.
(d) Tier progression.
(1) If any pathogenic or toxic effects are observed at the
maximum hazard dosage level in this study, testing at Tier II
[environmental expression testing ( 155A) is required as specified
in 40 CFR 158.740(d). In some cases, a subchronic test may serve
to better understand the effects observed at the Tier I level and
might alleviate the need for Tier II testing.
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134 (2) If no pathogenic effects are observed in this study, no
additional testing at higher tiers ordinarily is required. The
Agency nay require additional testing, however, if it determines
that there is a potential risk to birds despite the negative Tier
I results.
(e) References. The following references are provided for
use in the development of test protocols for conducting an avian
inhalation pathogenicity test with microbial pest control agents:
(1) Friend, M. and D.O. Trainer 1970. Polychlorinated biphenyl:
interaction with duck hepatitis virus. Science
170(3964):1314-1316.
(2) Friend, M. 1971. Experimental duck virus hepatitis
in the mallard. Avian Disease 16(4): 692-699.
(3) Friend, M. 1972. Duck hepatitis virus interaction with
DDT and Dieldrin in adult mallards. Bull. Environ. Oontam. Toxicol.
7(4):202-206.
(4) Friend, M. 1974a. Experimental DDT-Duck hepatitis virus
interaction studies. J. Wildl. Manage. 38(4): 887-895
(5) Friend, M. 1974b. Experimental Dieldrin-Duck hepatitis
virus interaction studies. J. Wildl. Manage. 38(4):896-904.
(6) Hartmann, G.C. and S.S. Wasti. 1980. Avian safety of
three species of entomogenous fungi. Camp. Physiol. Ecol. 5(4):
242-245.
(7) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
1975. Pp. 179-184. ijQ Guidelines for Safety Testing of Baculoviruses.
Baculoviruses for Insect Pest Control: Safety Considerations. American
Society for Microbiology Washington, D.C.
154A-18 Wild mammal toxicity and pathoqenicity testing;
Tier I.
(a) When required. Data on wild mammal toxicity and pathogen-
icity may be required by 40 CFR 158.740 (d) on a case-by-case basis
to support the registration of end-use formulated products (EP)
intended for outdoor application and manufacturing-use products
(MP) that legally may be used to formulate such end-use products.
The toxicity and pathogenicity data required by the toxicology
section of this subdivision for evaluating hazard to humans and
domestic animals are normally adequate to indicate potential hazard
to wild mammals. Under certain conditions, however, these data are
not sufficient to assess the potential hazard to wild mammals likely
to be exposed to a MPCA. An example of one circumstance when such
additional testing be required is the situation in which data indicate
that there is considerable variation in sensitivity of different
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Pesticide Assessment Guidelines
mammalian species to the effects of a MPCA agent, and there is
evidence that wild mammals will be heavily exposed to a MPCA. See
40 CFR 158.50 and 158.740(d) to determine whether these data must
be submitted.
(b) lest standards. Data must be derived from tests that
satisfy the general test standards in 150A-3 of this subdivision
and the following test standards;
(1) Test substance. The actual form of the material to be
regarded as the test substance is described in Section 150A-2(c) (2)
of this document. In addition, any substances used to enhance
virulence or toxicity should be tested along with the test substance.
(2) Species. Testing shall be performed on a mammalian
species representative or indicative of those found in the area(s)
likely to be affected by the proposed use pattern(s). Test animals
may be reared in pens or captured in the wild, and must be pheno-
typically indistinguishable from wild mammals. Endangered or
threatened animals shall not be used.
(3) Controls.
(i) A negative control group is required.
(ii) A concurrent control group is required and shall be
treated, when possible, with the pure active ingredient that has
been inactivated in such a way as to preserve cellular integrity.
(4) Route of exposure. The test material should be administered
by gavage (acute oral dose) or by intranasal instillation. The
method of dosing should reflect the expected exposure route and
shall be determined after consultation with the Agency.
(5) Maximum hazard dosage level. The standards for maximum
hazard dosage level, determination of an U^Q or !DKQ and duration
of test that are found in the avian oral pathogenicity/toxicity
test 154A-16 and the avian inhalation pathogenicity test 154A-17.
(c) Reporting and evaluation of data. In addition to the
information specified in 150A-4 of this subdivision, test reports
shall contain the same information required for the avian oral
pathogenicity/toxicity test 154A-16 and the avian inhalation patho-
genicity test 154A-17, adapted appropriately for mammalian test
procedures.
(d) Tier progression.
(1) If any toxic or pathogenic effects on mammalian species
are observed at the maximum hazard dosage level in this study,
testing at Tier II [environmental expression testing ( 155A) is
required as specified in 40 CFR 158.740(d). In some cases, a
subchronic test may serve to better understand the effects observed
at the Tier I level and might alleviate the need for Tier II testing.
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Subdivision M
(2) If toxic or pathogenic effects are not observed in this
study, additional testing at higher tiers ordinarily is not required.
The Agency may require additional testing, however, if it determines
that there is a potential risk to mammals despite negative Tier I
results.
(e) References. The following references are provided for
use in the development of test protocols for conducting wild mammal
toxicity and pathogenicity tests with microbial pest control agents:
(1) Barnes, R.W., C.F. Meincke, W.C. McLane, and C.S. Rehnborg.
1970. Lang-term feeding and other tcxicitypathogenicity studies on
rats using a commercial preparation of the nuclear-polyhedrosis
virus of Heliothis zea. J.. Invert. Pathol. 16:112-115.
(2) Fisher, R., and L. Rosner. 1959. Toxicology of the
microbial insecticide, Thuricide. Agricultural and Food Chemistry
7(10):686-688.
(3) Ignoffo, C.M., and A.M. Heimpel. 1965. The nuclear
polyhedrosis virus of Heliothis zea (Boddie) and Heliothis virescens
(Fabricius) Part V. Toxicity-pathogenicity of virus to white mice
and guinnea pigs. J.. Invert. Pathol. 7:329-340.
(4) Ignoffo, C.M. 1971. Intraperitoneal injection of white
mice with nucleopolyhedrosis virus of the beet armyworm, Spodoptera
exiqua. J. Invert. Pathol. 17(3):453-454.
(5) Ignoffo, C.M. 1973. Effects of entomopathogens on
vertebrates. Annals N.Y. Acad. Sci. 217:141-164.
(6) Ignoffo, C.M., J.J. Petersen, H.C. Chapman, and J.F.
Novotny. 1974. Lack of susceptibility of mice and rats to the
mosquito nematode, Reesimermis nielseni. Tsai and Grundmann. Mosquito
News 34(4):425-428.
(7) Ignoffo, C.M., C. Garcia, R.W. Kapp, and W.B. Coate.
1975. An evaluation of the risks to mammals of the use of an ento-
mopathogenic fungus, Nomuraea rileyi, as a microbial insecticide.
In: Baculoviruses for Insect Pest Control: Safety Considerations.
Selected papers from EPA/USDA. Working Symposium, Amer. Soc. Microb.,
Washington, D.C.
(8) Lamanna, C., and L. Jones. 1963. Lethality for mice of
vegetative and spore forms of Bacillus cereus and Bacillus cereus-
like insect pathogens injected intraperitoneally and subcutaneously.
J. Bacteriology 85:532-535.
(9) lautenschlager, R.A., C.H. Kircher, and J.D. Podgwaite.
1977. Effect of nucleopolyhedrosis virus on selected mammalian
predators of the gypsy moth. USDA, For. Serv. Res. Paper, NE-377,
6p.
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pesticide Assessment Guidelines
(10) Lautenschlager, R.A., and J.D. Podgwaite. 1979. Passage
of nucleqpolyhedrosis virus by avian and mammalian predators of the
gypsy moth, Lvmantria dispar. Environ. Entomol. 8(2) :210-214.
(11) Lautenschlager, R.A., and J.D. Podgwaite. 1977. Passage
of infectious nuclear-polyhedrosis virus through the alimentary
tracts of two small mammal predators of the gypsy moth, Lvmantria
dispar. Environ. Entomol. 6(5):737-738.
(12) Meinacke, C.F., W.C. Mclane, and C.S. Rehnborg. 1970.
Toxicity-pathogenicity studies of a nuclear-polyhedrosis virus of
Heliothis zea in white mice. J. Invert. Pathol. 15:10-14.
(13) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
1975. Pp. 179-184 in Guidelines for Safety Testing of Baculoviruses
Baculoviruses for Insect Pest Control: Safety Considerations. American
Society for Microbiology Washington, D.C.
(14) Watts, D.M., R.F. Tammariello, J.M. Dalrymple, B.F.
Eldridge, P.K. Russell, and F.H. Top, Jr. 1979. Experimental
infection of vertebrates of the Pocomoke Cypress Swamp, Maryland
with Keystone and Jamestown Canyon viruses. Am. J. Trop. Med.
Hvcr. 28 (2): 344-350.
(15) Wolf, K. 1975. Evaluation of the exposure of fish and
wildlife to nuclear polyhedrosis and granulosis viruses. Pp. 109-111
in Baculoviruses for Insect Pest Control: Safety Considerations.
American Society for Microbiology, Washington, D.C.
154A-19 Freshwater fish toxlcity and pathogenicity testing:
Tier I.
(a) When required. Data on pathogenicity and/or toxcity to
fish are required by 40 CFR 158.740(d) to support the registration
of each end-use formulated product (EP) intended for outdoor application
and each manufacturing-use product (MP) that legally may be used to
formulate such an end-use product. See 40 CFR 158.50 and 158.740(d)
to determine whether these data must be submitted.
(b) Test standards. Data must be derived from tests that
satisfy the general test standards in 150A-3 of this subdivision,
and the following test standards:
(1) Test substance. The actual form of the material to be
considered as the test substance is described in Section 154A-2(c) (2)
of this document. In addition, any substance used to enhance virulence
or toxicity should be tested along with the test substance.
(2) Test organisms.
(i) Testing shall be performed on one fish species, preferable the
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Subdivision M
rainbow trout if the MPCA has only a terrestrial use and direct aquatic
exposure is not expected or two fish species, preferably the bluegill
sunfish and rainbow trout when direct aquatic exposure is anticipated.
Other species of fish may be used, but a justification must be
supplied based on an increased susceptibility to the MPCA. or eco-
logical considerations that preclude the use of recommended species.
(ii) Ihe following characteristics should guide species selection:
(A) Fish species likely to prey upon or scavenge the target
host organisms should be tested, when applicable.
(B) Testing of young fish is preferable. Very young (not yet
actively feeding), spawning, or recently spent fish should not be
used.
(C) Fish should weigh between 0.5 and 5.0 grams and be from
the same year class. The length of the longest fish should be no
more than twice that of the shortest fish.
(iii) Ten fish per group should be used in multiple group
testing, thirty fish in single group testing.
(3) Route of Exposure.
(i) The test substance shall be administered as a suspension
directly into the water (i.e., aqueous exposure).
(ii) Additionally, the MPCA should be administered through
the oral route of exposure, preferably through incorporation in
standard fish food or through the use of infected insects.
(4) Maximum Hazard Dose. At a minimum, the concentration in
the test water (for aqueous exposure) should, whenever possible, be
at least 106 units/ml of water or at least 1000 times the maximum
calculated pesticide concentration in a six-inch layer of water
immediately following a direct application to a six-inch layer of
water, whichever is greater and attainable. Measures should be
taken to insure that the initial concentration of the MPCA is main
tained throughout the test should be described.
Feed used in the dietary exposure should be supplemented with
the test substance to achieve a microbial concentration of at least
100 times the calculated cell density in a 6 inch layer of water
immediately following a direct application to a 6 inch layer of
water.
(5) Controls.
(i) A negative, nondosed control group should be run concurrently
with the test groups.
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Pesticide Assessment Guidelines
(ii) A control group in which the fish are exposed to sterile
filtrate from production cultures should be performed concurrently
with the test groups.
(6) Test duration. The fish should be observed for a minimum
of 30 days after dosing. If symptomatology is present at the thirtieth
day, observation should be continued until recovery, mortality, or
unequivocal moribundity is established.
(7) Treatment concentrations.
(i) If the MPCA produces a toxin, then a sufficient number of
treatment concentrations must be tested to determine toxicity as
described in paragraph (b) (8).
(ii) If the MPCA does not produce a toxin, or no toxin has
been identified, then a single group of fish may be tested at the
maximum hazard dose. If deleterious effects, due either to toxicity
or pathogenicity are observed, sequentially lower doses should be
tested as described in paragraph (b) (8).
(8) Determination of LC50 or ID50. The study endpoint must
be chosen to reflect the activity of the specific microorganism
under test, i.e. if an MPCA is expected to produce a toxin and has
little or no infectivity/ then the appropriate endpoint would be
mortality. If, however, the major mechanism is pathogenicity, then
a more appropriate endpoint would be overt symptomatology.
The data should establish that the freshwater fish 1/250,
defined as the dose required to kill 50 percent of the test organisms,
or 1050, defined as the dose necessary to produce overt symptomatology
in 50 percent of the test organisms is greater than the maximum
hazard dosage level. If the LC50 or IC50 is lower than the maximm
hazard dose then an LC50 or 1050 with confidence intervals should
be established.
(c) Reporting and evaluation of data. In addition to informa-
tion meeting the general reporting requirements of 150A-4 of this
subdivision, a report of the results of a fish toxicity and infectivity
test must include the following:
(1) LC50 or IC5Q determination, including all associated
parameters e.g. slope, goodness of fit etc., along with the raw
mortality data.
(2) Detailed description of the steps taken to determine
microorganism dissemination, replication, or survival in any test
animals tissues, organs, or fluids:
(3) Detailed description of dilution water, including:
(i) Sterilization method;
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Subdivision M
(ii) Source;
(iii) Chemical characteristics (e.g., dissolved oxygen content,
pH, chlorine content, dissolved salts); and
(iv) Pretreatments (if any).
(4) Detailed description of the test, including:
(i) Design;
(ii) Container size;
(iii) Medium (e.g., depth and volume) ;
(iv) Prophylactic treatments;
(v) Number of organisms per treatment level;
(vi) loading (weight of organisms per unit volume of medium);
(vii) Lighting, acclimation, and test temperatures (averages
and range) ;
(viii) Amount of test substance administered by each route of
exposure; and
(ix) Any unusual feature of the test method.
(5) Detailed descriptions of methods (or references to estab-
lished methods) used for all chemical analyses of water for chemical
content and MPCA concentrations;
(6) Detailed description of methods used for all microbial
analyses of water, transport hosts and test organisms, and results
of such analyses.
(7) Detailed description of the effects of exposure to the
test substance including:
(i) The criteria used to determine the effects;
(ii) Percentages of test animals that died or showed symptomology;
and
(iii) A summary of these observations.
(8) Any additional relevant information about the test or its
results that would assisst in the determination of hazard potential.
(d) Tier progression.
(1) If toxic or pathogenic effects are observed, then testing
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pesticide Assessment Guidelines
at Tier II [environmental egression testing ( 155A) is required
as specified in 40 CFR 158.740(d). In some cases, a subchronic
test may serve to better understand the effects observed at the
Tier I level and might alleviate the need for Tier II testing.
(2) Further testing generally is not required if results of
this study do not indicate toxic or pathogenic effects. The Agency
may require additional testing if it determines that there is a
potential risk to fish despite negative Tier I results.
(e) References. The following may contain useful background
information for developing test protocols:
(1) Anonymous. 1975. Standard Methods for Examination of
Water and Wastewater. 14th Ed. American Public Health Assoc.,
Washington, D.C. 1193 pp.
(2) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians.
American Society for Testing and Materials, 1916 Race Street, Phila-
delphia, PA 19103.
(3) Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. U.S. Environmental Protection
Agency, Ecol. Res. Series, EPA 660/3-75-009. 61 pp.
(4) Hetrick, F.M., M.D. Khittel, and J.L. Fryer. 1979.
Increased susceptibility of rainbow trout to infectious hematopoietic
necrosis virus after exposure to copper. Appl. and Envir. Microb.
37(2):198-201.
(5) Huang, E., and J.S. Pagano. 1977. Nucleic acid hybridization
technology and detection of proviral genomes. Chapter 13 in The
Atlas of Insect and Plant Viruses, K. Maramorosch, Ed. Academic
Press, New York.
(6) Ignoffo, C.M. et al. 1973. Susceptibility of aquatic
vertebrates and invertebrates to the infective stage of the mosquito
nematode Reesimermis nielseni. Mosquito News 33(4): 599-602.
(7) Tatner, M.F., C.M. Johnson, and M.J. Home. 1984. The
tissue localization of Aeromonas salmonicida in rainbow trout,
Salmo gairdneri Richardson, following three methods of administration.
J. Fish Biol. 25: 95-108.
154A-20 Freshwater aquatic invertebrate toxicity and pathocrenicitv
testing; Tier I.
(a) When required. Data on pathogenicity and/or toxicity to
freshwater aquatic invertebrates are required by 40 CFR 158.740 to
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Subdivision M
support the registration of each end-use formulated product (EP)
intended for outdoor application and each manufacturing-use product
(MP) that legally may be used to formulate such an end-use product.
See 40 CFR 158.50 and 158.740(d) to determine whether these data
must be submitted.
(b) Test standards. Data must be derived from tests that
satisfy the general test standards in 150A-3 of this subdivision
and the following test standards:
(1) Test substance. The actual form of the material to be
regarded as the test substance is described in Section 154A-2(c) (2)
of this document. In addition, any substances used to enhance the
virulence or toxicity of the MPCA should be tested along with the
test substance.
(2) Test organisms.
(i) For MPCAs having terrestrial use patterns, where direct
aquatic exposure is not expected, one species of benthic invertebrate
should be tested. For MPCAs where direct aquatic exposure is anti-
cipated, testing shall be performed on two aquatic invertebrate
species, one of which is planktonic and the other benthic.
(ii) The species of aquatic invertebrate selected should bear
as close a taxonomic relationship to the target host as possible.
(iii) Aquatic invertebrate species likely to prey upon or
scavenge the diseased target host organisms should be tested, when
applicable.
(iv) Larval stages of invertebrates should be used
whenever possible.
(v) Twent invertebrates/group should be used if there are
multiple test groups. Fifty invertegrates should be used for single
group testing.
(3) Controls.
(i) A negative, nondosed control group should be performed
concurrently with the test groups.
(ii) A control group in which the invertebrates are exposed to
sterile filtrate from production cultures should be performed
concurrently with the test groups.
(4) Method of pesticide administration.
(i) The test substance shall be administered as a suspension
directly into the water (i.e., aqueous exposure).
(5) Maximum hazard dose. At a minimum, the concentration in
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pesticide Assessment Guidelines
the test water (for aqueous exposure) should, whenever possible, be
at least 106 units per ml of water or at least 1000 times the maximum
calculated pesticide concentration in a six-inch layer of water,
immediately following a direct application to a six-inch layer of
water, whichever is greater and attainable. The initial concentration
of MPCA. should be maintained throughout the test and the method
used for maintainence of the dose level should be described.
(6) Test duration. The test duration should be at least 21
days. If pathogenicity and/or toxicity are apparent at the
twenty-first day, observation should continue until recovery, morta-
lity, or unequivocal moribundity is established.
(7) Treatment concentrations.
(i) If the test substance produces a toxin, then a sufficient
number of treatment concentrations must be tested to permit a
determination of toxicity as described in paragraphs (b) (8).
(ii) If the test substance does not produce a toxin, or no
toxin has been identified, then a single group may be tested at the
maximum hazard concentration. If deleterious effects, due either to
toxicity or pathogenicity, are observed, then sequentially lower
doses should be tested as described in paragraph (b) (8).
(8) Determination of EC50 or ID50.
(i) Satisfactory data must establish whether or not the test
substance is pathogenic to the test organisms during a sufficiently
long period of exposure and observation.
(ii) If the test substance produces a toxin, then the data
must establish either:
(A) A definitive EC50 value with 95% confidence intervals; or
(B) That the EC50 is greater than the highest dose.
(c) Reporting and evaluation of data. In addition to information
meeting the general reporting requirements of 150A-4 of this sub-
division, a report of the results of an aquatic invertebrate toxicity
and infectivity test must include:
(1) Paw data and EC50 calculations including 95% confidence
intervals;
(2) A detailed description of the steps taken to determine
microorganism dissemination, replication, or survival in the test
animal tissues, organs, or fluids.
(3) A detailed description of dilution water, including
source, chemical characteristics (e.g., dissolved oxygen content,
pH, dissolved salts), method of sterilization, and pretreatment (if
any).
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Subdivision M
(4) A detailed description of the test, including:
(i) Design;
(ii) Container size;
(iii) Medium (e.g., depth and volume);
(iv) Pretreatments, if any;
(v) Method of exposing organisms to the test substance;
(vi) Number of organisms per treatment;
(vii) Lighting, acclimation, and test temperatures (averages
and range);
(viii) Amount of test substance administered; and
(ix) Any unusual feature of the test method.
(5) Detailed descriptions of methods (or references to estab-
lished methods) used for chemical analyses of water for chemical
content and MPCA concentrations.
(6) Detailed descriptions of methods used for all microbial
analyses of water and test organisms, and the results of such
analyses.
(7) Detailed description of the effects of exposure to the
test substance, including:
(i) The criteria used to determine the effects;
(ii) Statement of percentages of organisms that died or showed
effects of treatment; and
(iii) A summary of these observations, including changes in life
cycle (duration, fecundity/ and morphology).
(8) Any additional relevant information about the test or its
results that would assist in the determination of hazard potential.
(d) Tier progression.
(1) If toxic or pathogenic effects are observed, then testing
at Tier II [environmental expression ( 155A) shall be required.
In some cases, a subchronic test may serve to better understand the
effects observed at the Tier I level and might alleviate the need
for Tier II testing.
(2) If no toxic or pathogenic effects are observed, then no
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pesticide Assessment Guidelines
further testing at higher tiers ordinarily is required, except as
noted in paragraph (d) (3) of this section.
(3) If host spectrum or beneficial insect tests indicate a
broad host spectrum such that hazard toward aquatic invertebrates
is indicated/ then either:
(i) Additional aquatic invertebrate species must be tested as
described in paragraphs (a) through (c) of this section; or
(ii) Testing at Tier II, environmental expression ( 155A) is
required.
(4) If toxic or pathogenic effects are observed in tests
conducted in accordance with paragraph (d) (3) (i) of this section,
then testing at Tier II [environmental expression ( 155A) is required.
If not, then no further tier testing is ordinarily required. The
Agency may require additional testing, however, if it determines
that there is a potential risk to aquatic invertebrates despite
negative Tier I results.
(e) References. The following references may contain useful
background information for developing test protocols:
(1) Anonymous. 1975. Standard Methods for Examination of
Water and Wastewater. 14th Ed. American Public Health Assoc.,
Washington, D.C. 1193 pp.
(2) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians.
American Society for Testing and Materials, 1916 Race Street,
Philadelphia, PA 19103.
(3) Committee on Methods for Toxicity Tests with Aquatic
Organisms. Methods for Acute Toxicity Tests with Fish, Macro-
invertebrates, and Amphibians. U.S. Environmental Protection Agency,
Ecol. Res. Series, EPA 660/375-009. 61 pp.
(4) Huang, E., and J.S. Pagano. 1977. Nucleic acid hybridi-
zation technology and detection of proviral genomes. Chapter 13 in
The Atlas of Insect and Plant Viruses, K. Maramorosch, ed. Academic
Press, N.Y.
(5) Ignoffo, C. M. et al. 1973. Susceptibility of aquatic
vertebrates and invertebrates to the infective stage of the mosquito
nematode, Reesimermis nielseni. Mosquito News 33(4):599-602.
(6) Lightner, D.V., R.R. Proctor, A.K. Sparks, J.R. Adams,
and A.M. Heimpel. 1973. Testing Penaeid shrimp for susceptibility
to an insect Nuclear Polyhedrosis virus. Environ. Entomology 2(4):611-
613.
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Subdivision M
(7) Pagaho, J.S., and E. Huang. 1974. The application of
RNA-DNA cytohybridization to viral diagnostics. In: Viral Unrauno-
diagnosis. E. Kurstak and R. Mbrisset, eds. Academic Press, Inc.
New York.
(8) Reynolds, G.J. 1978. Enzyme labelled antibody in histo-
pathology. Qualitvline (Winter 1978/1979) :2-10.
(9) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
1975. Baculoviruses for Insect Pest Control: Safety Considerations.
Selected papers from EPAHJSDA Working Symposium, American Society
for Microbiology Washington, D.C.
(10) Uhdeen, A.H., and J.V. Maddox, 1973. The infection of
nomosquito hosts by injection with spores of the microsporidan
Nosema algerae. J. Invert. Path. 22:258-265.
(11) Van Essen, F.W., and D.W. Anthony. 1976. Susceptibility
of nontarget organisms to Nosema algerae (Microsporida: Nosematidae),
a parasite of mosquitoes. J.. Invert. Path. 28:77-85.
(12) Weber, C.E. (ed.) 1973. Biological field laboratory
methods for measuring the quality of surface waters and effluents.
U.S. Environmental Protection Agency, Environ. Monit. Series,
EPA-670/473-001.
154A-21 Estnarine and marine animal toxicity and
pathogenicitv tests; Tier I.
(a) When yeqm-npd. Data on pathogenicity and/or toxicity to
estuarine and marine animals are required by 40 CFR 158.740(d) to
support the registration of each end-use formulated product (EP)
and of each manufacturing-use product (MP) that legally may be used
to formulate such an end-use product for direct application into
the estuarine or marine environment or expected to enter this envi-
ronment in significant concentrations due to proposed use or mobility
pattern. See 40 CFR 158.50 and 158.740(d) to determine whether
these data must be submitted.
(b) Test standards. Data must be derived from tests that
satisfy the general test standards in 150A-3 of this subdivision
and the following test standards:
(1) Test substance. The actual form of the material to be
regarded as the test material is described in Section 154A-2(c) (2)
of this document. In addition, any substances used to enhance viru-
lence or toxicity should be included.
(2) Test organisms.
(i) Toxicity and pathogenicity shall be determined for one
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pesticide Assessment Guidelines
species of shrimp, preferably Paleomonetes vulqaris and one estuarine
or marine fish species.
(ii) Testing of additional estuarine or marine animal species
may be required in Tier I as specified in paragraph (d) (3) (i) of
this section.
(iii) Estuarine or marine animals likely to prey upon or
scavenge the diseased target host organisms should be tested, when
applicable.
(iv) Tests should be conducted using young fish, from the
samp year class, weighing between 0.5 and 5.0 grams. Very young
(not yet actively feeding), spawning, or recently spawned fish
should not be tested. The length of the largest fish should be no
more than twice that of the shortest fish.
(3) Controls.
(i) A negative, nondosed control should be performed concurrently
with the test groups.
(ii) A control group in which the animals are exposed to the
sterile filtrate from the manufacturing-use product should be per-
formed concurrently with the test groups.
(4) Method of pesticide administration.
(i) For fish, the MPCA wil be administered as a suspension
directly into the water and, separately, incorporated into the
food.
(ii) For shrimp, the MPCA will be incorporated into food pellets
which are then fed to the test organisms.
(5) Maximum Hazard Dose. At a minimum, the concentration in
the test water (for aqueous exposure) should, whenever possible, be
at least 106 untis/ml or at least 1000 tinreg the maximum calculated
cell density in a six-inch layer of water immediately following a
direct application to a six-inch layer of water, whichever is greater
and attainable.
Feed used in the dietary exposure should be supplemented with
the test substance to achieve a microbial concentration at least
100 times the calculated cell density in a 6-inch layer of water
immediately following a direct application to a 6-inch layer of
water.
(6) Test duration. The test should last at least thirty days.
If pathogenicity and/or toxicity is apparent at the thirtieth day,
observations should continue until recovery, mortality, or unequivocal
moribundity is established.
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Subdivision M
(7) Number of organisms per concentration. The number of test
organisms per group should be ten for fish and thirty for shrimp.
(8) Treatment concentrations.
(i) If the test substance produces a toxin, then a sufficient
number of treatment concentrations most be tested to determine
toxicity as described in paragraphs (b) (9) (ii) of this section.
(ii) If the test substance does not produce a toxin, or if no
toxin has been identified, then a single group may may be tested at
the maximum hazard dose. If deleterious effects, due either to
toxicity or pathogenicity, are observed, sequentially lower doses
should be tested as described in paragraph (b) (9).
(9) Determination of IC50 or ID50.
(i) Satisfactory data must establish an IC50 with 95% confidence
limits or, that the IC50 is greater than the highest dose.
(ii) If the test substance produces a toxin, then the data
must establish either:
(A) A precise LC50 or value with 95% confidence intervals; or
(B) That the IC^Q is greater than the highest dose.
(c) Reporting and evaluation of data. In addition to information
meeting the general requirements of 150A-4 of this subdivision, a
report of the results of estuarine or marine animal toxicity and
pathogenicity tests must include the following:
(1) LC50 data (if the test substance produces a toxin).
(2) A detailed description of the steps taken to determine
microorganism dissemination, replication or survival in the test
animal tissues, organs, or fluids;
(3) Detailed description of dilution water, including source,
chemical characteristics (e.g., dissolved oxygen content, pH, chlorine
content, dissolved salts), method of sterilization, and pretreatment
(if any);
(4) Other pertinent details, including;
(i) Design;
(ii) Container size;
(iii) Medium (e.g., depth and volume);
(iv) Pretreatments, if any;
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pesticide Assessment Guidelines
(v) Method of exposing organisms to the test substance (e.g.,
placing test substance in water which contains organisms or placing
organisms in water which contains the test substance);
(vi) Number of organisms per treatment;
(vii) Loading (weight of organisms per unit volume of medium
or unit of surface) ;
(viii) Lighting;
(ix) Acclimation and test temperatures (average and range);
(x) Salinities;
(xi) Amount of test substance administered;
(xii) Any unusual feature of the test;
(5) Detailed description of methods (or references to established
methods) used for all chemical analyses of water for chemical content
and MPCA concentrations;
(6) Detailed description of methods used in all microbial
analyses of water and test organisms, and the results of such analyses.
(7) Detailed description of the effects of exposure to the
test substance, including:
(i) The criteria used to determine the effects;
(ii) A statement of the percentage of organisms that died or
showed effects from the treatment; and
(iii) A summary of these observations.
(8) Any additional relevant information about the test or its
results that would assist in the determination of hazard potential.
(d) Tier progression.
(1) If toxic or pathogenic effects are observed, then testing
at Tier II [environmental expression ( 155A) is required as specified
in 40 CFR 158.740(d). In some cases, a subchronic test may serve
to better understand the effects observed at the Tier I level and
might alleviate the need for Tier II testing.
(2) If no toxic or pathogenic effects are observed, then no
further testing at higher tiers is ordinarily required, except as
noted in paragraph (d) (3) of this section.
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Subdivision M
(3) If efficacy or beneficial insect tests indicate a broad
host spectrum such that susceptability of estuarine or marine inver-
tebrates is indicated, then either:
(i) Additional estuarine or marine invertebrate species must
be tested following the guidelines in paragraphs (a) through (c) of
this section; or
(ii) Testing at Tier II [environmental expression ( 155A)
is required as specified in 40 CFR 158.740(d).
(4) If toxic or pathogenic effects are observed in tests
conducted in accordance with the requirements of this section, then
testing at Tier II [environmental expression ( 155A) is required.
Otherwise, no further tier testing is required.
(e) References. The following may contain useful background
information for developing test protocols:
(1) Anonymous. 1975. Standard Methods for Examination of
Water and Wastewater. 14th Ed. American Public Health Assoc.,
Washington, D.C. 1193 pp.
(2) ASTM Standard E 729-80, Practice for Conducting Static
Acute Toxicity Tests with larvae of Four Species of Bivalve Molluscs.
American Society for Testing and Materials, 1916 Race Street, Phila-
delphia, PA 19103.
(3) Anonymous. 1978. Bioassay Procedures for the Ocean
Disposal Permit Program. U.S. Environmental Protection Agency,
Office of Research and Development. EPA-600/9-78-010. 121 pp.
(4) Bahner, L.H., C.D. Craft, and D.R. Niramo. 1975. A
salt-water flow-through bioassay method with controlled temperature
and salinity. Proa. Fish-Cult. 37(3):126-129.
(5) Clark, J.R., and R. L. Clark, eds. 1964. Seawater
systems for experimental aquariums. U.S. Int. Dept. Fish, and
Wild. Serv., Bur. Sport. Fish. Wild. Res. Rep. No. 63, 192 pp.
(6) Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. U.S. Environmental Protection
Agency, Ecol. Res. Series, EPA 660/3-75-009. 61 pp. (Marine and
estuarine species listed in this publication are acceptable.)
(7) Couch, J.A., M.D. Summers, and L. Courtney. 1975.
Environmental significance of baculovirus infections in estu-
arine and marine shrimp. Annals N.Y. Acad. Sci. 219:528-536.
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pesticide Assessment Guidelines
(8) Couch, J.A. 1984. Design and Test of a Simple System for
the Preliminary Evaluation of Infectivity and Pathogenesis of Insect
Virus in a Nontarget Estuarine Shrimp. J. Invert. Path. 43: 351-357.
(9) DeBen, E.A. 1970. Design and construction of saltwater
environment simulator. Fed. Water Qual. Admin., Pacific N.W. Water
Lab., Working Paper 71:1-30.
(10) Hetrick, F.M., M.D. Khittel and J.L. Fryer. 1979.
Increased susceptibility of rainbow trout to infectious hematopoetic
necrosis virus after exposure to copper. Appl. Environ. Micro.
37(2):198-201.
(11) Huang, E. and J.S. Pagano. 1977. Nucleic acid hybridi-
zation technology and detection of proviral genomes. Chapter 13 in
The Atlas of Insect and Plant Viruses. K. Maramorosch, ed. Academic
Press, N.Y.
(12) Ignoffo, C.M., et al. 1973. Susceptibility of aquatic
vertebrates and invertebrates to the infective stage of the mosquito
nematode, Reesimermis nielseni. Mosquito News 33(4):599-602.
(13) Lightner, D.V., R.R. Proctor, A.K. Sparks, J.R. Adams,
and A.M. Heimpel. 1973. Testing Penaeid shrimp for susceptibility
to an insect Nuclear Polyhedrosis virus. Environ. Entomology 2(4):
611-613.
(14) Pagano, J.S., and E. Huang, 1974. The application of
RNA-DNA cytohybridization to viral diagnostics. In: Viral Immuno-
diagnosis. E. Kurstak. and R. Morisset, eds., Academic Press, Inc.,
N.Y.
(15) Reynolds, G.J. 1978. Enzyme labelled antibody in histo-
pathology. QualityliJie (Winter 1978/1979): 2-10.
(16) Shelbourne, H.E. 1962. Experimental seawater systems
for rearing fish larvae. Pp.81-93 in Seawater Systems for Experi-
mental Aquariums. J.R. Clark and R.L. Clark, eds. U.S. Dept.
Int., Fish. Wild. Serv., Bur. Sport Fish. Wild. Res. Rep. No.63.
192 pp.
(17) Strickland, J.D.H., and T.R. Parsons. 1968. A practical
handbook of seawater analysis. Fish Res. Board Can. Bull. No.
167., 311 pp.
(18) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
1975. Baculoviruses for Insect Pest Control: Safety Considerations.
Selected papers from EPA-USDA. Working Symposium, American Society
for Microbiology Washington, D.C.
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Subdivision M
(19) Tatner, M.F., C.M. Johnson, and M. J. Home. 1984. The
tissue localization of Aeromonas salmonicida in rainbow trout,
Salmo qairdneri Richardson, following three methods of administration.
J. Fish Biol. 25: 95-108.
(20) Undeen, A.H., and J.V. Maddox. 1973. The infection of
nomosquito hosts by injection with spores of the microsporidan
Nosema alaerae. J.. Invert. Path. 22:258-265.
(21) Van Essen, F.W., and D.W. Anthony. 1976. Susceptibility
of nontarget organisms to Nosema algerae (Microsporida: Nosematidae),
a parasite of Mosquitoes. J.. Invert. Pathology 28:77-85.
(22) Weber, C.E. (ed.) 1973. Biological field laboratory
methods for measuring the quality of surface waters and effluents.
U.S. Environmental Protection Agency, Environ. Monit. Series, EPA-
670/4-73-001.
154A-22 Plant studies; Tier I.
(a) When required.
(1) General. Data on the toxic or other adverse effects of a
pesticide organism on plant growth and development are required by
40 CFR 158.740 to support the registration of each end-use product
intended for outdoor application and each manufacturing-use product
that legally may be used to formulate such an end-use product. See
40 CFR 158.50 and 158.740 to determine whether these data must be
submitted.
(b) Test standards. In addition to satisfying the applicable
general test standards outlined in 150A-3 of this subdivision, this
study should meet the following standards:
(1) Test substance. The actual form of the material to be
regarded as the test substance is discussed in Section 154A-2 (c) (2)
of this document. In addition, any substances used to enhance
virulence should be tested along with the test substance.
(2) Dose levels. One concentration level equal to no less
than the maximum label rate shall be tested. The phrase "the
maximum label rate" means the amount of active ingredient in the
recommended quantity of carrier, such as water to be used per land
area or applied directly to the surface of a 15-cm or 6-in column
of water.
(3) Test species. The number of species tested depends
on the similarity of the MPCA to known plant pathogens. A rationale
for selection of the species to be tested must be provided to the
EPA.
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pesticide Assessment Guidelines
(i) Animal-controlling MPCAs. When the pesticide is intended
to control animals, including insects, the plants to be tested
should include six species of Dicotyledoneae of at least four
families and four species of Monocotvledoneae of at least 2 families.
These species should be selected from the plants of most important
commercial value (Table 3).
For MPCAs that have aquatic uses or may be expected to disseminate
to, and survive in, aquatic ecosystems, additional aquatic plants must
be tested to include: Selenastrum capricornutum (a freshwater green
alga), Lemna gibba (duckweed), Skeletonema costatum (a marine diatom),
Anabaena flos-aquae (a blue-green bacterium), and a freshwater
diatom.
EPA will consider requests for waiver of part or all of this
requirement if it can be shown that the MPCA occurs naturally in
the area of intended usage and the level used does not exceed the
naturally occurring concentration.
(ii) Plant-controlling MPCAs and MPCAs similar to known plant
pathogens. When the pesticide is intended to control plant growth
and development (microbial herbicides), or is otherwise closely
related to a plant pathogen, in addition to testing the range of
plants identified for animal-controlling MPCAs, testing must be
performed on all plants of economic importance (horticultural/
agronomic) or known to be beneficial to maintenance of the ecosystem
that have any reasonable likelihood of serving as hosts. This
selection of additional plant species should be based upon (1) a
survey of plants closely related (same genus or, if not available,
same family) to the target plant and (2) a survey of known hosts of
pathogens closely related to the microbial herbicide.
(4) Controls. Both positive and negative controls should be
included in the test protocols.
(i) Negative (untreated) controls should be as pest free as
reasonably possible. In addition, in the case of MPCAs that are
readily disseminated (wind, insects, etc.), it may be necessary to
conduct tests such that negative controls and treated plants are
grown in separate geographic locations or in separate contained
greenhouses under identical environmental conditions so that reliable
negative controls can be maintained. Alternatively, the negative
control may be treated with a nonphytotoxic chemical pesticide
known to provide effective control of the microbial pesticide.
Since it is sometimes difficult to detect adverse effects, such
as delayed maturation or loss in vigor, growth, quality, yield, or
stand, it is important to analyze untreated controls using a sensi-
tive, specific analytical method to determine whether or not MPCA
infection has occurred.
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Subdivision M
(ii) Positive controls are required for microbial herbicides,
or for MPCAs similar to known plant pathogens, in order to ascertain
that environmental conditions are such that penetration, infection,
and disease development are likely to occur in a susceptible host.
The positve control should be selected to closely resemble the sub-
ject MPCA in terms of taxonomy and optimal conditions for infection
and disease development, if known. In the case of a MPCA not in-
tended for herbicidal use, the positive control may consist of a
known plant pathogen, with taxonomic characteristics similar to the
MPCA, and its susceptible host. In the case of a microbial herbicide,
however, the positive control should consist of the target pest weed
and the microbial herbicide.
(5) Environmental Test Conditions. When the optimum conditions
for penetration, infection, and disease development are known or sus-
pected, it is important, particularly for microbial herbicides, to
simulate these conditions rather than those known to be optimum for
plant growth and development. In many cases, however, the optimum
environment may be similar.
(6) Application of MPCA. The test plants should be exposed
to the MPCA by whatever route of exposure would be expected by the
proposed use pattern. This route of exposure should be supple-
mented by other routes of exposure if indicated by the mode of
transmission of typical pathogens of the test plant or, for microbial
herbicides, if indicated by the mode of transmission of similar
plant pathogens. In some cases, wounding of plants or simulation
of (or actual) insect vectors might be appropriate. In other cases,
seed treatment, root (soil) application, or foliar spray might be
the most appropriate method.
(7) Timing of application. Plant test species should be
treated at the time of most likely susceptibility (this may be
known for microbial herbicides) or at the normal stage of maturity
when application to target areas is initiated.
(8) Observations. Plants should be observed weekly or more
frequently until normal harvest or death, or, in the case of peren-
nials, at regular intervals for at least 2 years. If no obvious
adverse effects are evident after these observation periods, the
roots, foliage, fruit, vascular tissues, etc. should be analyzed
for the presence of the organism using sensitive, specific methods.
It is important to complete such analyses because (i) obvious disease
development in perennials may take several years and (ii) asymptomatic
plants may serve as sites for proliferation and survival of the orga-
nism, thus providing a reservoir of the organism in the environment.
(c) Reporting. In addition to the information specified in
150A-4 of this subdivision, the test report shall contain the
following information.
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(1) Rationale for selection of the species tested.
(2) Description of the growth chambers, greenhouse, or other
type of test facility including containment provisions and monitoring
devices for maintaining proper environmental conditions.
(3) Temperature and humidity ranges, including any significant
deviations encountered in course of the experiment.
(4) Riotoperiod and lighting.
(5) Any abnormal adverse or beneficial effects in treatment and/or
control groups, including dates and times the effects were observed.
(6) Methods for any statistics used for analysis of results.
(d) Tier progression. If any adverse effects resulting from
infection occur or analyses indicate that asymptomatic infection
has occurred, testing at Tier II (Environmental Expression, 155A)
is required as specified in 40 CFR 158.740.
In some cases, a subchronic test may serve to better understand the
effects observed at the Tier I level and might alleviate the need
for Tier II testing.
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146
Table 3-25 Major Agricultural Crops
Listed by * Value
Corn
Soybeans
Wheat
Cotton
Tobacco
Sorghum
Potatoes
Oranges
Bar ley
Rice
Grapes
Peanuts
Apples
Sugarbeets
Sugarcane
Tomatoes
Lettuce
Oats
Strawberr ies
Beans
Onions
Peaches
Almonds
Sunflower
Broccoli
Listed by Ibs Produced
Corn
Alfalfa/Hay
Wheat
Soybeans
Sorghum
Sugarcane
Sugarbeets
Potatoes
Oats
Oranges
Grapes
Apples
Cotton
Grapefruit
Peanuts
Plums
Sunf1ower
Barley
Peaches
Tobacco
Beans
Cucumbers
Rice
Rye
Peas
NOTE: Depending upon the predicted use pattern, certain forest
tree species, ornamental trees and shrubs, and weed species may
need testing.
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Pesticide Assessment Guidelines
154A-23 Nontarcret insect testing for toxicity/pathogenicitv to insect
predators and parasites: Tier I.
(a) When -regmnpH. Data on the toxicity/pathogenicity of a
microbial pest control agent are required Toy 40 CFR 158.740 to
support the registration of each end-use product containing a
microbial pest control agent intended for outdoor application
when the proposed use pattern indicates that insect predators
and/or parasites may be exposed to the pesticide, and each
manufacturing use product that legally may be used to formulate
such an end-use product. See 40 CFR 158.740 to determine
whether these data must be submitted.
(b) Test standards. In addition to satisfying the applicable general
test standards outlined in 150A-3 of this subdivision, this
study should meet the following standards:
(1) Test substance. The actual form of the material to be regarded
as the test substance is discussed in Section 154A-2(c) (2) of
this document. In addition, any substances used to enhance
virulence should be tested along with the test substance.
Whenever feasible, dosage shall be in suitable increments up
to 100X the ID^Q or IC50 of the pathogen in its natural host,
or 10 to 100 tomes the recommended field dosage.
(2) Test species. Testing should be performed on three species of
insects, representing at least two of the following groups:
Parasitic dipterans
Predaceous hemipterans
Predaceous coleopterans
Predaceous mites
Predaceous neuropterans
Parasitic hymenopterans
Selection of test species is the responsiblity of the researcher.
Rationale for selection must be provided. More specifically, the
following points apply:
(i) Viruses.
Testing shall be performed on three species of insects
representative of those parasites and predators known or
suspected to attack the target host or to share the same
ecological habitat.
(ii) Protozoa.
In selecting the 3 species of non-target arthropods, at
least one should be a major (i.e. , important) parasite of
the target host. Many protozoans have a wide host range.
Accordingly, if possible, more than the minium™ (3) species
of non-target organisms should be tested.
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Subdivision M
(iii) Fungi.
Assuming testing is justified, appropriate organism test species
should be the major regulatory agents caramon to the ecosystem
where the MPCA will be applied. In addition, testing could
include parasites and/or predators specific to the host of the
fungal agent.
(iv) Bacteria. Testing shall be performed on three species of insects
representative of those parasites and predators known or sus-
pected to attack the target host or to share the same ecological
habitat.
(3) Controls. A concurrent control group is recommended
and should be treated with microbe-free (or non-viable
microbe) material from the culture system used for propagation
of the microbial pest control agent.
(4) Routes of exposure.
(i) Viruses.
The best routes of exposure will depend on the developmental
location of the nontarget organism. Internal stages may be
tested with virus-infected hosts, or if they are culturable
in vitro, the virus can be added to the diet. External
stages, if they are obligatory, may be fed virus-infected
hosts, and others may be fed virus-contaminated media or
virus suspended in sugar or honey solutions.
(ii) Protozoa.
In addition to feeding adult predators and parasites of the
target insect with the resistant stage of the protozoan,
immature stages of the predator or parasite should be
exposed. Predators can be fed hosts infected with the
protozoan over a period of time. The predator, at a
prescribed time, should be checked for protozoan infection.
The protocol for parasites is more complex. Protozoan-
infected hosts can be parasitized or parasitized hosts can
be fed protozoans. Parasites from protozoan-infected hosts
should be examined for protozoan infection. The immature
stages as well as adults should be examined. If possible,
a primary hymenopterous and dipterous parasite should be examined.
The best route of infection for adult Hymencptera or Diptera
is oral acquisition of protozoan spores. Predaceous insects
could also be fed in this manner, but feeding them infected
(live) hosts of known age is more appropriate. In either
case, the acutal amount of spores consumed cannot be
accurately determined. If infection of the parasite or
predator adult occurs, the possibility for transovarial or
transovum transmission should be examined.
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pesticide Assessment Guidelines
(iii) Funcri.
Routes of exposure should simulate field conditions as
much as possible. In the case of entomopathogenic fungi,
environmental conditions (>90% R.H.) are critical at the
time of exposure. No scientific data is available on
comparative exposure techniques.
(iv) Bacteria. Best routes of exposure will depend on developmental
location of the nontarget organism. Internal stages may be tested
with bacteria-infected hosts, or if they are culturable in vitro,
the bacteria can be addpd to the diet. External stages may be fed
bacteria-infected hosts, bacteria-contaminated media, or bacteria
suspended in sugar or honey solutions.
(5) Duration of test/endpoints.
(i) Viruses.
Control and treated insects should be observed for a duration
of at least 30 days after dosing, or in cases where an insect
species cannot be cultured for 30 days, until control mor-
tality rises above 20%. In cases where signs, symptoms and
pathologies are detected, the treated insects should be
examined in detail at late stages of infection, at moribund,
and at death. Such tests need not be prolonged to 30 days
if death of treated insects occurs prior to 30 days. In
all cases of pathologies in the treated nontarget insects,
it is essential that the etiology of the infection be
established.
The end-points should be based on the frank development of
pathologies and on the early mortality of the treated as
compared to the untreated (control) nontarget organisms.
(ii) Protozoa.
Test duration should be determined on a case-by-case basis.
The most appropriate end-point for protozoan diseases for
determining pathogenic effects is the presence of the
vegetative stages (shizonts or meronts) in the tissues of
non-target insects. The schizonts within suspected tissues
can be detected by making a smear, staining with Giemsa
stain, and examining the slide with oil immersion using a
compound microscope. The non-target insect should be alive
when the tissues are removed for the smear because the
shizonts are fragile and are usually destroyed by other
microbes or are distorted upon death of the host.
Protozoan spores can be used as an indicator of infection.
However,, if the infection is light, the few spores could
have come from the inoculum. If spores are abundant (a
relative term) and occur in the tissues of the nontarget
insect, it is likely that it is infected.
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Subdivision M
Another way to confirm infection is to conduct histological
studies of the tissues using standard methods and looking
for spores and other pathological effects. The end-point would
be just before death of the organism or a prescribed period
of time.
Death of the non-target insect is a good end-point if the
protozoan is virulent. However, since most protozoans have
chronic effects on their host, changes in behavior, size, or
color could be used as an end-point. In each case, a
microscopic examination to find schizonts or spores is
essential to confirm the presence of the protozoan. Koch's
postulates should be run.
(iii) Fungi.
With entomopathogenic fungi, test duration can be limited
to 8-10 days.
Mortality in time LT(50) is considered the most reliable
parameter for bioassaying fungi of insects in the laboratory.
Pathogenicity should be confirmed by identifying the fungal
agent as the original inoculum.
(iv) Bacteria.
Control and treated insects should be observed for 21 to 30
days after dosing, if this is possible. In cases where the
insect species cannot be cultured for 21 to 30 days, ob-
servation should continue until control mortality rises
above 20%.
(c) Reporting and evaluation of data. The reporting provisions are
the same as those specified in 150-4 of this subdivision.
(d) Tier progression.
(1) Data derived from Tier I testing will be used in conjunction
with available information on use pattern, host range, and
other similar factors, to assess potential for adverse effects.
If data indicate potential for adverse effects, Tier II testing
will be required as specified in 40 CFR 158.740. In some
cases, a subchronic test may serve to better understand the
effects observed at the Tier I level and might alleviate the
need for Tier II testing.
(2) If toxic or pathogenic effects are not observed in this study,
additional testing is ordinarily not necessary.
(e) References.
The following references are provided for use in the development
of acceptable test protocols for conducting toxicity/pathenogenicity
tests with microbial pesticides:
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pesticide Assessment Guidelines
Andreadis, T.G. 1980. Nosena pyrausta infection in Macrocentrus
orandii. a braconid parasite of the European corn borer, Ostrinia
nubilalis. J. Invertebr. Pathol. 35:229-233.
Brooks, W.M. 1973. Protozoa: Host - parasite - pathogen inter-
relationships. Misc. Pub. Entomol. Soc. Amer. 9:105-111.
Gardner, W.A., R.D. Getting, and G.K. Storey. 1982. Susceptibility
of the two-spotted spider mite, Tetranychus urticae Koch, to the
fungal pathogen Hirsutella thompsonii Fisher. Florida Entomol.
65(4): 458-465.
Van Essen, F.W., and D.W. Anthony. 1976. Susceptibility of nontarget
organisms to Nosema alaerae ( Microsporida: Nosematidae ), a parasite
of mosquitoes. J. Invert. Pathol. 28: 77-85.
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Subdivision M
154A-24 Honey bee.toxicitv/pathoaenicitv test; Tier I.
(a) When required. Data on the toxicity/pathogenicity of a microbial
pest control agent are required by 40 CFR 158.740 to support the regis-
tration of each end-use product intended for outdoor application when the
proposed use pattern indicates that honey bees may be exposed to the
pesticide, and for each manufacturing-use product that legally may be
used to formulate such an end-use product. See 40 CFR 158.50 and 158.740
to determine whether these data must be submitted.
(b) Test standards. In addition to satisfying the applicable general
test standards outlined in 150A-3 of this subdivision, this study shall
meet the following standards;
(1) Test substance. The actual form of the material to be regarded
as the test substance is discussed in Section 154A-2(c) (2) of this document.
In addition, any substances used to enhance virulence should be tested
along with the test substance.
(2) Test species. Testing shall be performed on the honey bee,
Apis mellifera.
(3) Age. Test insects should be worker bees of uniform age.
(4) Controls. A concurrent control group is recommended and should
be treated with microbe-free (or non-viable microbe) material from the
culture system used for propagation of the microbial pest control agent.
(5) Duration of test. Control and treated bees should be observed
for at least 30 days after dosing.
(c) Reporting and evaluation of data. The reporting requirements
are the same as those specified in 150A-4 of this subdivision.
(d) Tier progression.
(1) Data derived from Tier I testing will be used in conjunction
with available information on use pattern, host range, and other factors,
to assess potential for adverse effects. If data indicate that the poten-
tial for adverse effects exists, Tier II testing will be required as
specified in 40 CER 158.740. In some cases, a subchronic test may
serve to better understand the effects observed at the Tier I level and
might alleviate the need for Tier II testing.
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(2) If toxic or pathogenic effects are not observed in this study,
additional testing is ordinarily not necessary.
(e) References. The following references are provided for use in
the development of acceptable test protocols for conducting a honey bee
toxicity/pathogenicity test with a microbial pest control agent:
(1) Davidson, W.W., H.L. Morton, J.O. Mbffett, and S. Singer.
1977. Effect of Bacillus sphaericus strain SSII-1 on honey bees, Apis
mellifera. J. Invert. Pathol. 29:344-346.
(2) Menapace, D.M., R.R. Sackett, and W.T. Wilson. 1978. Adult
honey bees are not susceptible to infection by Nosema locustae. J. Econ.
Entomol. 71:304-306.
(3) Morton, H.L., J.O. Moffett, and F.D. Stewart. 1975. Effect of
alfalfa looper nuclear polyhedrosis virus in honey bees. J. Invert.
Pathol. 24:139-140.
(4) Hitchcock, J.D., A. Stoner, W.T. Wilson, and D.M. Menapace.
1979. Pathogenicity of Bacillus pulvifaciens to honey bee larvae of
various ages (Hymenoptera: Apidae)- J. Kansas Entomol. Soc. 52(2): 238-246.
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Subdivision M
TIER III TESTING.
154A-25 Terrestrial wildlife and aquatic organism toxicitv
testing; TJ^T III.
(a) When required. The data outlined in section series 71
and 72 of Subdivision E are required by 40 CFR 158.740 to support
the registration of each end-use product and each manufacturing-use
product that may be legally used to formulate such an end-use
product when toxic effects on nontarget terrestrial wildlife or
aquatic organisms are reported in one or more Tier I tests (154A-16
through -21) and results of Tier II tests (section series 155A)
indicate exposure of the MPCA to the affected nontarget terrestrial
wildlife or aquatic organisms. See 40 CER 158.50 and 158.740 to
determine whether these data must be submitted.
(b) Test standards. The test standards are the same as those
found in 71-1 through -5 and 72-1 through -6 of Subdivision E.
(c) Reporting and evaluation of data. The reporting and
evaluation provisions are the same as those found in 71-1
through -5 and 72-1 through -6 of Subdivision E.
(d) Tier progression. Further testing shall be required as
specified in 40 CFR 158.740 and outlined in 71-1 through -5 and
72-1 through -6 of Subdivision E.
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154A-26 Chronic avian pathoqenicitv and reproduction
test; Tier III.
(a) When required. Data on the chronic avian pathogenicity
and reproduction effects of a MPCA are required by 40 CER 158.740(d)
to support the registration of each end-use formulated product (EP)
intended for outdoor application and each manufacturing-use product
(MP) that legally may be used to formulate such an end-use product
when:
(i) Pathogenic effects are observed in Tier I ( 154A-16 and
-17) at any dose level and
(ii) Environmental expression testing ( 155A) indicates
that long-term exposure of terrestrial animals to low-level field
residues of the MPCA is likely. See 40 CFR 158.50 and 158.740(d)
to determine whether these data must be submitted.
The test is intended to determine if, when animals are exposed
to low levels of the MPCA over an extended period of time, chronic
effects will occur.
(b) Test standards. Data must satisfy the general
test standards in 150A-3 of this subdivision, and the following
test standards:
(1) Test substance. The actual form of the material to
be tested is described in Section 154A-2(c) (2) of this document. In
addition, any substances used to enhance the virulence should
be tested along with the test substance.
(2) Species. Testing shall be performed on one avian
species (preferably the bobwhite quail or mallard duck).
The species selected shall be the same as that used for the
avian inhalation pathogenicity test in 154A-13.
(3) Age. Birds approaching their first breeding season
shall be used.
(4) Controls. A concurrent control group is required
and shall be treated with inactivated MPCA.
(5) Treatment levels. At least two treatment levels
shall be used. The test concentrations should include an
actual or expected field residue exposure level and a multiple
of that level such as 5x.
(6) Dosing. The MPCA should be incorporated into the
diet whenever possible. The diet should be analyzed for
MPCA viability and new diet should be prepared often enough
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Subdivision M
so as to insure that the test animals receive a constant dose
throughout the study. Alternate routes of dosing, along with
the rationale, should be discussed with the Agency.
(7) Number of birds per treatment group. Each treat-
ment group should be replicated. For botawhite quail and
mallard ducks, a minimum of 12 pen replicates for each treat-
ment level should be used.
(8) Duration of exposure. Birds shall be exposed to
treated diets beginning not less than 10 weeks before egg
laying is expected, and extending throughout the laying
season.
(9) Duration of test. The test shall not end before
14 days after the last natchling leaves the shell.
(c) Reporting and evaluation of data. In addition to
the information specified in 150A-4 of this subdivision, the
test report shall contain the following information:
(1) Testing protocols. All methods used in performing
these tests should be fully referenced or described in detail.
(2) Test results. The following information shall be
reported for all test groups:
(i) Any observed abnormal behavior;
(ii) All results of tests used to detect the presence
of the MPCA;
(iii) Time and date of mortalities;
(vii) Results of gross necropsy tests conducted on all
birds dying or manifesting clinical symptomatology at test
termination. Necropsies should also be performed on at least
50 percent of the nonaffected birds.
(viii) Reisolation of microbes from selected body tissues
that would normally be affected by an infection including
the liver, kidney, lungs, spleen, cerebrospinal system, testes
ovaries, oviducts, and gastrointestinal tract and from all dead
embryos, and an assessment of the clinical significance of such
isolations;
(ix) Morbidity;
(x) Accidental deaths or injuries; and
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pesticide Assessment Guidelines
(xi) Observed clinical signs and symptoms
(3) Test conditions. The following information shall
be reported for treated and untreated test groups:
(i) Species;
(ii) Age;
(iii) Body weight;
(iv) Number and sex of birds in each treatment group;
(v) Individual identification of birds;
(vi) Diet composition and additives (especially
antibiotics;
(vii) Diet storage conditions and results of periodic
assays for MPCA content;
(viii) Feed consumption (grams per day);
(ix) Observation on palatability or repellency;
(x) Housing conditions of test birds:
(A) Space allocations for mating and nesting;
(B) Measures taken to insure that the birds were
protected from injuries;
(C) Lighting program, including hours per day and wat-
tage or footcandles at bird level;
(xii) Diagram of test layout:
(xiii) Temperature;
(xiv) Water source and its microbiological and chemical
characteristics if sterilized distil led water is not used; and
(xv) Pretest and test history of the test organisms
with respect to medical and chemical treatments.
(3) Egg and hatching data.
The following information shall be reported for each treatment
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Subdivision M
group:
(i) Eggs laid (nuniber eggs per bird per day and per
season);
(ii) Hatching egg storage data:
(A) Temperature;
(B) Humidity;
(C) Nuniber of eggs placed in incubator;
(D) Egg-turning frequency;
(iii) Number of embryonated eggs;
(iv) Number of embryos that hatch;
(v) Ratio of hatched embryos to the number placed in
the incubator;
(vi) Number of dead embryos;
(vii) Physical abnormalities in hatchlings;
(viii) All observed clinical signs and symptoms;
(ix) Post-hatchling mortality; and
(x) Weights of fourteen-day-old survivors.
(4) Pesticide test data. Ihe concentration of the
MPCA in the feed must be monitored weekly, more often if MPCA
viability is short, and the results tabulated. For large
quantities of treated feed, a statistically-sound sampling
method should be employed.
(d) Tier progression.
(1) If pathogenic and/or reproduction effects are observed
at actual or expected exposure levels:
(i) The applicant should reconsider the proposed regis-
tration of the product; and
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pesticide Assessment Guidelines
(ii) The Agency will, at this time, review all the data
and determine if a decision regarding acceptability for
registration should be made. Testing at Tier IV, simulated
or actual field testing ( 154A-33) may not be feasible if
adequate containment or quarantine methods are not possible.
If they are, testing at Tier IV is required as specified by
40 CER 158.740(d).
(2) If no pathogenic effects are observed at actual or
expected field residue exposure levels, no additional testing
is ordinarily required.
(e) References. The following references are provided
for use in the development of test protocols for conducting
chronic avian pathogenicity and reproduction tests with
microbial pest control agents:
(1) Heinz, G.H. 1976. Methylmercury: Second year feed-
ing effects on mallard reproduction and duckling behavior.
J. Wildl. Manaa. 40(1):82-90.
(2) Heinz, G.H., and L.N. Locke. 1976. Brain lesions
in mallard ducklings from parents fed nethylmercury. Avian
Diseases 20(1):9-17.
(3) U.S. Environmental Protection Agency, Registration
of Pesticides in the United States: Proposed Guidelines,
Subdivision E—Hazard Evaluation: Wildlife and Aquatic Organisms.
1978 (July 10). Fed. Reg. 43(132):29729-29731.
154A-27 Aquatic invertebrate range tegt-inpf* Tier HI.
(a) When required. Data from aquatic invertebrate
range testing are required by 40 CER 158.740(d) to support
the registration of each end-use formulated product (EP)
intended for use in water or expected to be transported to
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Subdivision M
water from the intended use site, and when pathogenicity or
infectivity was observed in Tier I tests on aquatic invertebrates.
These tests are required to support the registration of each
manufacturing-use product (MP) that legally may be used to
formulate such an end-use product. See 40 CER 158.50 and
158.740 (d) to determine whether these data must be submitted.
(b) Test standards. Data must be derived from tests
that satisfy the general test standards in 150A-3 of this
subdivision, and the test standards in Tier I ( 154A).
(1) Test organisms. In selecting invertebrate species
for this test, one should strive to choose species most likely
to be affected by the MPCA under test. Thus, if a microorganism
that is closely related (within the same family) to the MPCA
causes disease in a certain invertebrate species, that species
should be included in this test.
Use pattern also plays a role in selection. Microbial
pest control agents that are expected to enter freshwater
ecosystems should be tested on freshwater aquatic invertebrates.
Likewise, if an estuary is likely to be impacted, marine
invertebrates should be included.
In any case, testing should be performed on two members
of the following orders.
freshwater
Cladocera
Copepoda
Ephemeroptera
Trichoptera
Marine
Copepoda
Crustacea
(3) Method of pesticide administration. The test sub-
stance shall be administered either as a suspension in the
test water (aqueous exposure) and/or in the diet as determined
from results of Tier I tests.
(4) Dose levels. The dose level shall be equal to that
expected to be found in the aquatic environment calculated
from application rates with appropriate adjustments to take
into account the environmental survival and multiplication
characteristics, as determined by Tier II testing, of the
MPCA under test.
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Pesticide Assessment Guidelines
(5) Test duration. The test duration shall be the
same as Tier I studies.
(c) Reporting and evaluation of data. The requirements
in Tier I, 154A-16 and -17, apply.
(d) Tier progression.
(i) If pathogenic effects are observed, further testing
at Tier IV ( 154A-29) may be required.
(ii) If pathogenic effects are not observed, additional
testing at higher tiers ordinarily is not required.
(e) References. Refer to paragraph (e) in 154A-20
and -21.
154A-28 Fish life cycle studies; Tier III.
(a) When required. Data from fish life cycle studies
are required by 40 CFR 158.740(d) to support the registration
of each end-use formulated product (EP) intended for use in
water or expected to be transported to water from the intended
use site, and when pathogenicity or infectivity was observed
in Tier I tests. These tests are required to support the
registration of each manuf acturing-use product (MP) that may
be legally used to formulate such an end-use product. See
40 CFR 158.50 and 158.740(d) to determine whether these
data must be submitted.
(b) Test standards. Data must be derived from tests
that satisfy the general test standards in 150A-3 of this
subdivision, and the following test standards:
(1) Test substance. The actual form of the test material
to be used is described in Section 154A-2(c) (2) of this document.
In addition, any substances used to enhance virulence should
be tested along with the test substance.
(2) Duration of test. The fish life-cycle tests require
that the test animals be cultured in the presence of the
MPCA during one complete life cycle, i.e. one stage of the life
cycle to the same stage of the next generation.
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Subdivision M
(3) Test organisms. In selecting a species of fish for
this test, one should strive to choose the species most likely
to be affected by the MPCA under test. Thus, if a microorganism
that is closely related (within the same family) to the MPCA
causes disease in a certain fish species, that species should be
chosen as the test organism.
Use pattern also plays a role in the selection. Microbial
pest control agents that are expected to enter freshwater
ecosystems should be tested on freshwater fish. Likewise, if
an estuary is likely to be impacted, marine fish should be
used.
In the event that a species cannot be identified using
the foregoing criteria, suggested species are as follows.
(i) Freshwater species:
° Brook trout (Salvelinus fontinalis)
° Fathead minnow (Pimephales promelas)
° Bluegill sunfish (Lepomis marochirus)
° Channel catfish (Ictalurus punctatus)
(ii) Marine/Esturine species:
° Sheepshead minnow (Cyprinodon variegatus)
0 Atlantic silverside (Menidia menidia)
(4) Dose levels. The dose levels shall be equal to that
expected to be found in the aquatic environment calculated
from application rates with appropriate adjustments to take
into account the environmental survival and multiplication
characteristics, as determined by Tier II testing, of the MPCA
under test.
(5) Test procedure. Refer to the listed references for
general guidance in design of fish life cycle tests. The
applicant should consult with the Agency for testing details.
(6) Test duration. For test species with life cycles of
a year or less, the test should begin with newly-hatched
larvae and continue until thirty days after progeny hatch.
When fish with life cycles that are greater than one year,
begin the test with fish that are about to commence their
first spawn and continue until ninety days after the progeny
hatch.
(c) Reporting and evaluation of data. In addition to
the information specified in 150A-4 of this subdivision, the
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pesticide Assessment Guidelines
test report shall contain the following information on the
nontarget test organism:
(1) Reproductive effects;
(2) Effects on the growth of larvae and juveniles;
(3) Detailed records of spawning, egg numbers, fertility,
and fecundity/
(4) Estimated no observed effect level;
(5) Mortality data;
(6) Statistical evaluation of effects;
(7) Lcxxmotion, behavioral, physiological, and patho-
logical effects;
(8) Definition of the criteria used to determine effects;
(9) Summary of observed signs of pathogenicity or
other effects;
(10) Isolation, identification, and enumeration of
microorganism (s) responsible for any observed pathogenic
effects; and
(10) Stage of life cycle in which effects occurred.
(d) Tier progression.
(1) If pathogenic effects are observed, further testing
at Tier IV 154A-33 may be required.
(2) If pathogenic effects are observed, additional
testing at higher tiers ordinarily is required.
(e) References. The following may contain useful
background information for developing acceptable protocols:
(1) Fish life-cycle tests:
(i) Anonymous. 1985. Standard Methods for Examination
of Water and Wastewater. 16th Ed. American Public Health
Assoc., Washington, D.C. 1268 pp.
(ii) Biesinger, K.E. 1974(a). Procedure for Daphnia
magna tests in standing system. U.S. Environmental Protec-
tion Agency, Environ. Res. Lab., Duluth, Minn.
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Subdivision M
(iii) Biesinger, K.E. 1974 (b). Procedure for Dapihnia
maqna chronic tests in flowing system. U.S. Environmental
Protection Agency, Environ. Res. Lab., Duluth, Minn.
(iv) Hansen, D.J., P.R. Parrish, S.C. Schiiranel, and
L.R. Goodman. 1978. Life-cycle toxicity test losing sheeps-
head minnows (Cyprinodon varieqatus). Pp. 109-116 in Bioas-
say Procedures for the Ocean Disposal Permit Program. U.S.
Environmental Protection Agency, Office of Res. and Dev.
EPA-600/978-010.
(v) Nimmo, D.E., T.L. Hamaker, and C.A. Sommers. 1978.
Entire life-cycle toxicity test using mysids (Mysidopsis
bahia) in flowing water. Pp. 64-68 in Bioassay Procedures
for the Ocean Disposal Permit Program. U.S. Environmental
Protection Agency, Office of Res. and Dev. EPA-600/9-78-010.
(vi) Schiiranel, S.C., and D.J. Hansen, 1974. Sheepshead
minnow Cyprinodon varieqatus; an estuarine fish suitable for
chronic (entire life-cycle) bioassays. Proc. 28th Ann.
Cong. S.E. Assoc. Game-Fish Comm. Pp. 392-398.
(vii) National Water Quality Laboratory Committee on
Aquatic Bioassays. 1971. Recommended bioassay procedure
for fathead minnow Pimephales promelas (Rafinesqui) chronic
tests. National Water Quality Laboratory, Duluth, Minn. 13
pp. (Revised January 1972.)
(3) Additional information Additional information may
be found in the following reference:
(i) Biesinger, K.E. 1974 (c). Culturing methods for
Daphnia and certain other cladocerans. U.S. Environmental
Protection Agency, Environ. Res. Lab., Duluth, Minn.
154A-29 Aquatic ecosystem disruption studies; Tier III.
(a) When required. Data from aquatic ecosystem dis-
ruption studies are required by 40 CFR 158.740(d) to support
the registration of each end-use formulated product intended
for outdoor application and each manufacturing use product
that legally may be used to formulate such an end-use product.
If, after an analysis of the MPCA's ability to survive and
multiply in the environment and what ecological habitat it
will occupy, the intended use patterns, and the results of
previous nontarget organism and environmental expression
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tests, it is determined that use of the microbial agent may result
in adverse effects on the nontarget organisms in aquatic environ-
ments, then testing is required to determine if application of the
microbial pest control will be expected to disrupt the balance of
populations in the target ecosystem. See 40 CFR 158.50 and
158.740(d) to determine whether these data must be submitted.
(b) Test standards. Specific requirements will be established
on a case-by-case basis. Data sufficient to satisfy the general
test standards in 150A-3 of this subdivision, and the following
test standards:
(1) Test substance. Die actual form of the material
to be tested is described in Section 154A-2(c) (2) of this documaent.
in addition, any substances used to enhance virulence should
be tested along with the test substance.
(2) Test system. The microbial pest control agent will be
tested in a complex, realistic microcosm which should be designed
in consultation with the Agency. The microcosm should contain members
from all trophic levels found in natural aquatic ecosystems.
(2) Test organisms.
(i) Following consultation with the Agency, the registration
applicant shall choose a combination of the following species
that reflects the species diversity of the target ecosystem:
(A) A typical bottom-feeding fish (e.g., catfish or
carp);
(B) A cold-water fish, a warm-water fish, or a marine
fish (e.g., brook trout, rainbow trout, bass, bluegill,
northern pike, walleye, or sheepshead minnow);
(C) Molluscs (e.g., oyster or freshwater clams);
(D) Crustaceans (e.g., Daphnia spp., shriinp, or cray-
fish); or
(E) Nymphs (e.g., mayfly).
(c) Reporting and evaluation of data. In addition to
the information required by 150A-4 of this subdivision, specific
data reporting and evaluation requirements will be established
on a case-by-case basis following consultation with the
Agency.
(d) Tier progression.
(1) If pathogenic effects are observed then simulated and
actual field testing may be required.
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Subdivision M
(2) If pathogenic effects are observed, additional
testing at higher tiers ordinarily is required.
(e) References. The following nay contain useful
background information for developing acceptable protocols:
(1) Johnson, B.T., and R.A. Schoettger. 1975. A
biological model for estimating the uptake, transfer, and
degradation of xenobiotics in an aquatic food chain. Fed.
Regis. 40 (123):26906-26909. (June 25, 1975.)
(2) Macek, K.J., M.E. Barrows, R.F. Frasny, and B.H.
Sleight, III. 1975. Bioconcentration of CL4-pesticides
by bluegill sunfish during continuous exposure. Pp. 119-142
in Structure-activity correlations in studies of toxicity
and bioconcentration with aquatic organisms. 6.D. Veith and
D.E. Kbnasevich, eds. Proceedings of a Symposium, Burlington,
Ontario, March 11-13, 1975. Sponsored by Standing Committee
on Scientific Basis for Water Quality Criteria of the
International Joint Commission's Research Advisory Board.
(3) Schimmel, S.C., J.M. Patrick, Jr., and A.J. Wilson.
1977. Acute toxicity to and bioconcentration of endosulfan
by estuarine animals. Pp. 241-252 in Aquatic Toxicology and
Hazard Evaluation. F.L. Mayer and J.L. Hamelink, eds.
STP #634, American Society for Testing and Materials,
Philadelphia, Pennsylvania.
154A-30 Special aquatic tests - tissue culture, microorganism/
stress interaction testg. [Reserved]
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154A-31 Plant studies; Tier III.
(a) When required. Data on the effects of a MPCA on plant
growth and development are required by 40 CFR 158.740(d) to support
the registration of each end-use formulated product intended for
outdoor application and each manufacturing-use product that legally
may be used to formulate such an end-use product where the material
may transport from the site of application by air, soil, or water.
The extent of movement will be determined by the environmental
expression tests in Tier II ( 155A)
(b) Test standards. The test standards shall be developed
on consultation with the Agency. These tests will be designed to
analyse any significant adverse effects seen in Tier I tests. A
range of dose levels will be used in order to determine if the
population levels of the MPCA seen in the Tier II studies are able
to produce adverse effects. These Tier III protocols may incorporate
aspects of Tier IV studies in that the tests may be conducted under
simulated or actual environmental conditions.
(c) Reporting. The reporting requirements shall be the
same as those in 154A-22(c) of this Subdivision.
(d) Tier progression. If any adverse effects resulting from
infection occur or analyses indicate that asymptomatic infection
has occurred in Tier III tests, testing at Tier IV (Simulated or
Actual Field Testing, 154A-37) is required as specified in 40
CFR 158.740.
154A-32 [Reserved]
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Subdivision M
'I'l Kk IV TESTING
154A-33 Simulated or actual field testing for mammals
and birds; Tier IV.
This study is required on a case-by case basis when
deleterious effects are observed in Tier III testing. The
applicant should consult with the Agency for specific study
methodology.
154A-34 Simulated or actual field testing for aquatic
organisms! Ti^r IV.
This study is required on a case-by-case basis when
deleterious effects are observed in Tier III testing. The
applicant should consult with the Agency for specific study
methodology.
154A-35 Simulated or actual field testing for insect
predators and parasites; Tier IV
This study is required on a case-by-case basis when
deleterious effects are observed in Tier I testing. The
applicant should consult with the Agency for specific study
methodology.
154A-36 Simulated or actual field testing for insect
pollinators; Tier IV
This study will be required on a case-by-case basis when
deleterious effects are observed in Tier I testing. The
applicant should consult with the Agency for specific study
methodology.
154A-37 Simulated or actual field testing for plants; Tier IV
This study will be required on a case-by-case basis when
deleterious effects are observed in Tier III testing. The
applicant should consult with the Agency for specific study
methodology.
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Pesticide Assessment Guidelines
Series 155A: Tier II ENVIRONMENTAL EXPRESSION DATA
FOR MPCAs
155A-1 General Information
(a) Purpose. Tier II tests are intended to demonstrate whether a
MPCA is able to survive or replicate in the environment, thereby indicating
v*iich_nantarget_organism(s) may be exposed to the microbial agent, as
well as provide an indication of the level of exposure. A determination
of the environmental expression of a MPCA includes: an evaluation of the
growth of the agent when introduced into a new environment; the way the
MPCA may alter its growth habits, take advantage of new environmental
conditions or take advantage of changes in the existing equilibrium among
microbial species (e.g., in a commensal association, an association in
which one species benefits and the other is unaffected). Thus, the ex-
pression of a microorganism's presence may result in its insertion into a
new habitat and continued propagation in the new habitat.
Terrestrial and aquatic (freshwater and marine) environments are to
be considered, as indicated by results from Tier I, the product type and
the product usage. Advance consultation with Agency personnel is recom-
mended since requirements may be modified on a case-by-case basis.
The Tier II guidelines consist of descriptions of tests to determine the
environmental expression of a MPCA in a terrestrial environment
( 155A-10), in a fresh water environment ( 155-11) and in a marine environment
( 155A-12). If Tier II data is negative (no adverse effects), no further
testing is needed.
(b) When Required.
(1) General. Tier II environmental expression data are required by
40CFR 158.740 when toxic or pathogenic effects are observed in maximum
hazard testing conducted on nontarget organisms in Tier I tests ( 154-
16 through 24). Table 5 summarizes the relationship between Tier I test
results, product type, intended use patterns and the need for Tier II
testing. When no adverse effects are observed in Tier I, no Tier II
tests are required. Positive data from Tier II provide a basis for further
testing as described in Tiers III and IV. Tier II data will be used to
establish descriptions of conditions and doses to be used in subsequent
testing.
(2) Specific. Specific Tier II data are required when Tier I tests
demonstrate adverse effects. See 40 CER 158.50 and 158.740 to deter-
mine whether these data must be submitted.
(i) Terrestrial Environment. Tier II data on the expression of a
MPCA in a terrestrial environment are required by 40 CER 158.740 to
support the registration of each end-use formulated product intended for
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Subdivision M
outdoor application, on land and each manufacturing-use product that
legally may be used to formulate such an end-use product when adverse
effects (toxic or pathogenic) are observed in any of the following Tier I
tests:
(i) Avian oral pathogenicity/toxicity test: Tier I. ( 154A-16) ;
(ii) Avian respiratory pathogenicity test ( 154A-17);
(iii) Wild mammal toxicity and pathogenicity test ( 154A-18);
(iv) Plant studies - terrestrial ( 154A-22);
(v) Testing of toxicity/pathogenicity to insect predators
and parasites ( 154A-23); or
(vi) Honey bee toxicity/pathogenicity test ( 154A-24).
(2) Freshwater Environment. Tier II data on the expression of a
MPCA in a freshwater environment are required by 40 CFR 158.740 to
support the registration of each end-use formulated product intended for
outdoor application is applied to or may enter freshwater environments
and each manfucturing-use product that legally may be used to formulate
such an end-use product when adverse effects (toxic or pathogenic) are
observed in any of the following Tier I tests:
(i) Avian oral pathogenicity/toxicity test: Tier I. ( 154A-16);
(ii) Avian respiratory pathogenicity test ( 154A-17);
(iii) Wild mammal toxicity and pathogenicity test ( 154A-18) ;
(iv) Freshwater fish toxicity and pathogenicity testing ( 154A-19) ;
(v) Freshwater aquatic invertebrate toxicity and pathogenicity
tests ( 154A-20); or
(vi) Plant studies - aquatic ( 154A-22).
(3) Marine or Estuarine Environment. Tier II data on the expression
of a MPCA in a marine or estuarine environment are required by 40 CFR
158.740 to support the registration of each end-use formulated product
intended for outdoor application is applied to or may enter the marine
or estuarine environments and each manufacturing-use product that legally
may be used to formulate such an end-use product when adverse effects
(toxic or pathogenic) are observed in any of the following Tier I tests:
(i) Avian oral pathogenicity/toxicity test: Tier I. ( 154A-16) ;
(ii) Avian respiratory pathogenicity test ( 154A-17);
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Pesticide Assessment Guidelines
(iii) Estuarine and marine animal toxicity and pathogenicity test
( 154A-21); or
(iv) Plant studies - estuarine or marine ( 154A-22).
(c) Approach. Environmental expression testing consists of simulated
terrestrial and aquatic applications of the MPCA. Terrestrial applications
are conducted in a contained environment to assess expression in soil and
vegetation. Aquatic applications are conducted in aquaria to assess
expression in water and sediment. The need for terrestrial, freshwater
or marine environmental expression testing under specific conditions
depends on:
- The Tier I tests in which adverse effects were observed; and
- The intended use pattern (s) for the MPCA.
Testing is only required to assess environmental expression when
susceptible nontarget species may be exposed. Thus, Tier I tests define
the need and the environment to be examined. In some cases, no tests may
be needed. For example, if adverse effects are noted with terrestrial
plants and the intended use pattern calls for use in freshwater only,
probably no additional tests would be required. If additional tests are
required (e.g., adverse effects with terrestrial insects and a terrestrial
use pattern), the timing (e.g., season of use, time of day for release)
and location (e.g., climate, soil type) of usage, as well as doses shown
to be effective in Tier I, must be considered in selecting Tier II con-
ditions for establishing environmental expression of the test microbe.
This simulation of the particular natural environment reflecting the area
of use of the product is necessary in order to achieve the overall objective
of Tier II testing - determining whether the introduced microorganism will
survive, multiply and potentially threaten the nontarget species. The
intent is to define potential exposure of susceptible nontarget species
to the test product.
Thus, data should be developed which would indicate the dynamics of
population fluctuation (i.e. growth and/or survival curves) of the micro-
organism. The experimental design should consider parameters such as
inoculum size, potential for blooms or regrowth, and physical parameters
such as relative humidity, pH and temperature.
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172
Table 4 - Summary of Environmental Expression Testing as
Determined By Tier I Test Results and Use Patterns
Tier I Test with Positive
Results (Test Species)
Proposed Use Patterns for
MPCA
Terrestrial Freshwater Estuarine/Marine
154A-16 and 17
Avian testing - mallard
— quail
N/A1
T2
F3
N/A
EM4
N/A
154A-18
Mammalian testing
N/A
154A-19-21
Fish testing
- freshwater spp.
- estuarine/marine spp.
F
EM
F
EM
N/A
EM
154A-20 and 21
Aquatic invertebrate testing
- freshwater spp.
- estuarine/marine spp.
F
EM
F
EM
N/A
EM
154A-22
Terrestrial plant testing
N/A
N/A
154A-22
Aquatic plant testing
- freshwater spp. F
- estuarine/marine spp. EM
F
EM
N/A
EM
154A-23 and 24
Terrestrial insect testing
N/A
N/A
%/A: Not applicable - Based on results of Tier I bests and the proposed use
pattern, adverse effects are not expected. However, the Agency may
require such tests on an individual basis.
2T : Tests to determine expression in a terrestrial environment are necessary.
3F : Tests to determine expression in a freshwater environment are necessary.
4EM : Tests to determine expression in an estuarine or marine environment are
necessary.
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Pesticide Assessment Guidelines
(a) Applicability. Ihis section outlines the general test standards
that apply to the terrestrial, freshwater and aquatic studies.
Applicants for registration should also comply with the specific
test standards established for the particular test being conducted.
In the case of conflict between general and specific test standards,
the latter shall govern.
(b) Test standards. Data satisfying the provisions of 155A-2 should
meet the general test standards in Subdivision N ( 160-4 through-6),
with the following exception:
MPCA identification and ouantification. The most specific available
methods for the identification and quantification of the MPCA
should be used. The methods used should be consistent with those
in 150A-20, Product analysis.
(c) Definitions.
(1) Contained environment. Refers to the use of natural materials
from the proposed use-environment (sediment, soil, plants, and marine/
estuarine liquids) arranged as naturally as possible, and held within a
plastic, glass, or other container to prevent the escape of the microbial
agent.
(2) Substance to be tested. A typical end-use product (EP) or the
technical grade of the active ingredient (TGAI) shall be tested.
(3) Observation period. This should be of sufficient duration to un-
equivocally determine survival, growth or decline (to the limit of detection)
of the MPCA in the appropriate environment. Decline of the
MPCA should be followed for a sufficient period of time to detect
regrowth (see (4) below) of the agent after a prolonged adaptation period.
(4) Reqrowth. The number of microorganisms present may decrease
initially (perhaps to an undectable level) and then increase (regrowth) to
or above the initial cell density. Observations should continue for a
period of time sufficient to assure lack of regrowth. In addition, efforts
to resuscitate the MPCA (such as enrichment techniques and/or passage in
susceptible hosts) should be attempted to support the conclusion that the
microbial agent no longer persists in the environment.
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Subdivision M
155A-2 Reporting and evaluation of data.
(a) Results.
(1) Data collected to determine whether the MPCA is able
to survive, persist or replicate in the environment under Tier II test
conditions is best expressed in the form of a population growth or decline
curve for the MPCA, although other applicable methods of presenting
the data may be used.
(2) Test reports should also contain the information designated in
the following list, modified as necessary to be applicable to the microbial
agent and environment being tested. This information should be given in
sufficient detail to adequately define potential impact on growth character-
istics of the test organism. In general, the amount of information and
kind of data that should be reported in Tier II testing are very similar
for any of the environments being examined and are, where applicable, as
follows:
(i) type of system and components used;
(ii) source of components;
(iii) equilibrium period before addition of MPCA
(iv) temperature, light requirements, humidity or moisture content,
pH of the medium, oxygen requirements;
(v) type of formulation (EP or TGAI);
(vi) application rate or dose level;
(vii) identity of test substance and composition of formulation;
(viii) characteristics of water in aquatic systems;
(ix) method of application;
(x) sampling amount, schedule, and sensitivity of detection;
(xi) tabulation/graphs of population dynamics;
(xii) discussion of test results.
______ If the results of Tier II expression testing in
simulated, contained environments show that the MPCA will sur-
vive and persist in the environment under study, thereby exposing non-
target organisms to the MPCA, then further testing on the
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Pesticide Assessment Guidelines
Tier III level is required. If the results are negative (the microbial
agent does not survive or persist), then no further testing is required.
Unequivocal determination of negative results may be difficult to prove.
Diligent efforts using enrichment techniques may be needed to prove the
results are negative and that progression to Tier III is not required.
155A-2 Through 152A-9 [Reserved]
155A-10 Tests to determine expression in a terrestrial environment.
(a) When required. Data on the egression of a microbial pest con-
trol agent in a terrestrial environment are required by 40 CER 158.740 to
support the registration of each end-use product intended for outdoor
application on land and each manufacturinguse product that legally may be
used to formulate such an end-use product when toxic or pathogenic effects
are observed in any of the following Tier I tests:
(1) Avian oral pathogenicity/toxicity test: Tier I. ( 154A-16) ;
(2) Avian respiratory pathogenicity test ( 154A-17) ;
(3) Wild mammal toxicity and pathogenicity test ( 154A-18) ;
(4) Plant studies - terrestrial ( 154A-22);
(5) Testing of toxicity/pathogenicity to insect predators
and parasites ( 154A-23); or
(6) Honey bee toxicity/pathogenicity test ( 154A-24).
(7) See 40 CFR 158.50 and 158.740 to determine whether these
data must be submitted.
(b) Test standards.
(1) Method.
(i) Tests shall be conducted in a greenhouse environment to
determine whether the MPCA is able to survive, persist, and replicate
in a terrestrial environment consisting of soil and vegetation
representative of the proposed use site. The following parameters
should be varied to determine their effect on the survival and
growth of the MPCA population:
(A) Temperature;
(B) Humidity;
(C) Precipitation (amount, frequency, pH);
(D) Sunlight;
(E) pH (soil and foliar surfaces); and
(F) Nutrients (soil, vegetation).
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Subdivision M
(ii) The values selected for each parameter listed in paragraph
(b) (1) (A) through (F) of this section should be selected to approxi-
mate the conditions expected at the intended use site.
(iii) Laboratory studies designed to determine the microbial
agent's growth requirements (e.g., temperature, humidity, pH, sun-
light, and nutrients) may supplement the greenhouse study described
in paragraph (b) (1) (i). Laboratory studies may demonstrate that
the MPCA will be unable to survive and the Agency will
consider studies on a case-by-case basis to meet the intent of
testing in 155-18 in lieu of the greenhouse study.
(2) Test substance. A typical end-use product or the tech-
nical grade of the active ingredient shall be tested.
(3) Test duration. Data to establish a population decline
curve shall be collected at intervals until two half-life deter-
minations have been made or until data establish that the microbial
agent population is able to maintain itself in the terrestrial
environment at or above the level present immediately after test
initiation.
(c) Reporting and evaluation of data. The reporting and
evaluation provisions are the same as those set forth in 155A-2.
(d) Tier progression. If results of this study indicate that
the MPCA is able to persist in the terrestrial environment
such that the susceptible nontarget organism(s) tested in Tier I are
likely to be exposed, then the appropriate testing in Tier III
(154A-25, -26, or -31) is required as specified in 40 CER 158.740.
(e) References. The following references contain information
for developing acceptable protocols:
(1) Anthony, D.W.; Savage, K.E.; Hazard, E.I.; Avery, S.W.;
Boston, M.D.; Oldacre, S.W. (1978) Field test with Nosema alqerae
Vavra and Uhdeen (Microsporida, Nosematidae) against Anopheles
albimanus Wledemann in Panama. Miscel. Publ. Entomol. Soc. Amer.
11:17-28.
(2) Armstrong, J.L.; Knudsen, G.R.; Seidler, R.L. (1987)
A microcosm method to assess survival of recombinant bacteria as-
sociated with plants and herbivorus insects. Current Microbiol.
in press.
(3) CXinningham, J.C. (1970) Persistence of the nuclear
polydrosis virus of the eastern hemlock looper on balsam foliage.
Insect Pathology Res. Institute. Sault Ste. Marie, Ontario, Canada.
Bimonthly Res. Notes 26:24-25.
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Pesticide Assessment Guidelines
(4) Elgee, E. (1975) Persistence of a virus of the white-marked
tussock moth on balsam fir foliage. Maritime Forest Res. Centre.
Fredericton, New Brunswick, Canada. Bimonthly Res. Notes 31:33-34.
(5) Grison, P.; Martouret, D.; Servais, B.; Devriendt, M.
(1976) Microbial pesticides and environment. Ann. Zool. Ecol. Anim.
8(2):133-160.
(6) Harcourt, D.J. (1968) Persistence of a granulosis virus
of Pieris rapae in soil. J. Invert. Path. 11:142-143.
(7) Hukuhara, T.; Namura, H. (1972) Distribution of a
nuclear polyhedrosis virus of the fall webworm Hvphantria cunea in
soil. J. Invert. Path. 19:308-316.
(8) Ignoffo, C.M.; Garcia, C.; Hostetter, D.L.; Pinell, R.E.
(1978) Stability of conidia of an entomopathogenic fungus Nomuraea
rileyi in and on soil. Environ. Entomol. 7(5):724-727.
(9) Ignoffo, C.M.; Garcia, G.; Hostetter, D.L.; Pinnell, R.E.
(1977) Vertical movement of conidia of Nomuraea rilevi through sand
and loam soils. Environ. Entomol. 7(2):270-272.
(10) Jaques, R.P. (1967b) The persistence of a nuclear polyhe-
drosis virus in the habitat of the host insect Trichoplusia ni 11.
Polyhedra in soil. Can. Entomol. 99:820-829.
(11) Jaques, R.P. (1969) Leaching of the nuclear polyhedrosis
virus of Trichoplusia ni from soil. J. Invert. Path. 13:256-263.
(12) Jaques, R.P. (1974a) Occurrence and accumulation of
viruses of Trichoplusia ni in treated field plots. J. Invert. Path.
23:140-152.
(13) Jaques, R.P. (1974b) Occurrence and accumulation of the
granulosis virus of Pieris rapae in treated field plots. J. Invert.
Path. 23:351-359.
(14) Kerr, A. (1974) Soil microbiological studies on
Agrobacterium radiobacter and biological control of crown gall.
Soil Sci. 118(3):168-172.
(15) Ladd, Jr. T.L.; McCabe, P.J. (1967) Persistence of
spores of Bacillus popilliae, the causal organism of Type A milky
disease of Japanese beetle larvae in New Jersey soils. J. Econ.
Entomol. 60(2):493-495.
(16) Liang, L.N.; Sinclair, J.; Mallory, L.; Alexander, M.
(1982) Fate in model ecosystems of microbial species of potential
use in genetic engineering. Appl. Environ. Microbiol. 44:708-714.
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178
Subdivision M
(17) Lingg, A.J.; McMahon, K.J. (1969) Survival of
lyophilized Bacillus popilliae in soil. Appl. Microbiol. 17:718-720.
(18) Milner, R. J.; lutton, G.G. (1976) Metarrhizium
anisopliae; Survival of Oonidia in the Soil. Proceedings of the
First International Colloquium on Invertebrate Pathology. Queen's
University at Kingston, Canada. Pages 428-429.
(19) Morris, O.N. (1973) A method of visualizing and assessing
deposits of aerially sprayed insect microbes. J. Invert. Path.
22:115-121.
(20) Narayanan, K.; Govindarajan, K.; Jayarai, S. (1977)
Preliminary observations on the persistence of nuclear polyhedrosis
virus of Spodoptera litura F. Madras Agric. J. 64(7):487-488.
(21) Pinnock, D.E.; Brand, R.J.; Milstead, J.E.; Jackson, K.L.
(1975) Effect of tree species on the coverage and field persistence
of Bacillus thurinqiensis spores. Insect biological control.
J. Invert. Path. 25(2):209-214.
(22) Roone, R.E.; Daoust, R.A. (1976) Survival of the
nuclear polyhedrosis virus of Heliothis armiaera on crops and in soil
in Botswana. J. Invert. Path. 27:7-12.
(23) Swift, M.J. (1982) Microbial succession during the decay
of organic matter. In: Burns, R.G.; Slater, J.H. (eds) Experimental
microbial ecology. Oxford: Blackwell, 164-177.
(24) Thomas, E.D.; Reichelderfer, C.F.; Heimpel, A.M. (1973)
Ihe effect of soil pH of cabbage looper nuclear polyhedrosis virus
in soil. J. Invert. Path. 21(l):21-25.
(25) Vankova, J.; Svestka, M. (1976) The field persistence
and efficacy of Bacillus thurincriensis formulations. Biological
control of forest pests. Anz Schadlingskd Pflanzenschutz. 49(3):33-
38. (English Summary)
(26) Walter, M.V.; Barbour, K.; McDowell, M.; Seidler, R.J.
(1987) A method to evaluate survival of genetically engineered
microorganisms in soil extracts. Current Microbiol. In Press.
(27) Walter, M.V.; Porteous, A.; Seidler, R.J. (1987) Mea-
suring genetic stability in bacteria of potential use in genetic
engineering. Appl. Enviorn. Microbiol. 53:105-109.
(28) Wojciechlowska, M.; Rnitowa, K.; Fedorko, A.; Bajan, C.
(1977) Duration of activity of entomopathogenic inicroorganisms
introduced into the soil. Pol. Ecol. Stud. 3(2): 141-148.
(29) Young, S.Y. (1975) Pre- and post-treatment assessment
of virus levels. Pages 139-142 In selected papers from EPA-USDA
Working Symposium. M. Summers, R. Engler, L. Falcon, and P. Vail
(eds.) American Society of Microbiology, Washington, D.C.
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Pesticide Assessment Guidelines
155A-11 Tests to determine expression in a freshwater environment.
(a) When required.
(1) Data on the expression of a microbial pest control agent
in a freshwater environment are required by 40 CFR 158.740 to support
the registration of each end-use product intended for outdoor application
on land and each manufacturing-use product that legally may be used
to formulate such an end-use product when toxic or pathogenic effects
are observed in any of the following Tier I tests:
(i) Freshwater fish toxicity and pathogenicity test (154A-19);
(ii) Freshwater aquatic invertebrate toxicity and pathogenicity
test (154A-20); or
(iii) Plant studies - aquatic (154A-22).
(2) Data on the expression of a MPCA in a freshwater environ-
ment are required by 40 CER 158.740 to support the registration of
each end-use product intended for outdoor application on fresh
water and each manufacturing-use product that legally may be used
to formulate such an end-use product when toxic or pathogenic effects
are observed in any of the following Tier I tests:
(i) Avian oral pathogenicity/toxicity test: Tier I. ( 154A-16) ;
(ii) Avian respiratory pathogenicity test ( 154A-17) ;
(iii) Wild mammal toxicity and pathogenicity test ( 154A-18) ;
(iv) Freshwater fish toxicity and pathogenicity testing ( 154A-19) ;
(v) Freshwater aquatic invertebrate toxicity and pathogenicity
tests ( 154A-20); or
(vi) Plant studies - aquatic ( 154A-22).
(3) See 40 CFR 158.50 and 158.740 to determine whether
these data must be submitted.
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Subdivision M
(b) Test standards.
(1) Method.
(i) Tests shall be conducted in a simulated aquatic environment
(e.g., aquarium with bottom sediment) to determine whether the MPCA
is able to survive, persist, and replicate in a freshwater environment
consisting of fresh water and bottom sediment representative of the
proposed use site. The following parameters should be varied to
determine their effect on the survival and growth of the MPCA popu-
lation:
(A) Temperature;
(B) pH;
(C) Nutrients;
(D) Sunlight;
(E) Oxygen content;
(F) Hardness; and
(G) Turbulence.
(ii) The values selected for each parameter listed in
paragraph (b) (1) (A) through (G) of this section should be selected
to approximate the conditions expected at the intended use site.
(iii) Specialized laboratory studies designed to determine
the MPCA's growth requirements (e.g., temperature, pH,
sunlight, and oxygen) may supplement the study described in paragraph
(b) (1) (i) of this section. Specialized lab studies may demonstrate
that the MPCA will be unable to survive and persist in a
freshwater environment. In such instances, the Agency will consider
studies on an individual basis to meet the intent of testing in
155A-11 in lieu of the study described in paragraph (b) (1) (i) of
this section.
(2) Test substance. A typical end-use product or the technical
grade of the active ingredient shall be tested.
(3) Test duration. Data to establish a population decline
curve should be collected at intervals until two half-life determi-
nations have been made or until data establish that the microbial
agent population is able to maintain itself in a freshwater environ-
ment at or above the level present immediately after test initiation.
(c) Reporting and evaluation of data. The reporting and
evaluation provisions are the same as those set forth in 155A-2.
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Pesticide Assessment Guidelines
(d) Tier progression. If results of this study and use
patterns information indicate that the MPCA is likely to
enter and is able to persist in a freshwater environment such that
the susceptible nontarget organism (s) tested in Tier I are likely
to be exposed, then the appropriate testing in Tier III (154A-25
through -31) is required as specified in 40 CFR 158.740.
(e) References. The following references contain useful
information for developing acceptable protocols:
(1) Anonymous. (1975) Impact of the use of microorganisms on
the aquatic environment. EPA publication 660-3-75-001. Technical
Publications Office, Environmental Protection Agency, National
Environmental Res. Center, Corvallis, Oregon 97330.
(2) Anthony, D.W.; Savage, K.E.; Hazard, E.I.; Avery, S.W. ;
Boston, M.D.; Oldacre, S.W. (1978) Field tests with Nosema alaerae
Vavra and Uhdeen (Microsporida, Nosematidae) against Anopheles
albinamus Wiedemann in Panama. Misc. Publ. Entomol. See. Amer.
11:17-28.
(3) Brand, R.J.; Pinnock, D.E.; Jackson, K.L.; Milstead, J.E.
(1975) Methods for assessing field persistence of Bacillus thurin-
giensis spores. J. Invert. Path. 25:199-208.
(4) Hostetter, D.L.; Ignoffo, C.M.; Kearby, W.H. (1975)
Persistence of formulations of Bacillus thurinqiensis spores and
crystals on eastern red cedar foliage in Missouri. J. Kansas
Entomol. Sec. 48(2):189-193.
(5) Ignoffo, C.N.; Hostetter, D.L.; Pinnell, R.E. (1974)
Stability of Bacillus thurinqiensis and Baculovi TIIS heliothis on
soybean foliage. Environ. Entomol. 3(1):117-119.
(6) Kaya, H.K. (1975) Persistence of spores of Pleistophora
schuber (Onidospora: Microsporida) in the field and their application
in microbial control. J. Invert. Path. 26:329-332.
(7) Pinnock, D.E.; Brand, R.J.; Milstead, J.E. (1971) The
field persistence of Bacillus thurinqiensis spores. J. Invert. Path.
18:405-411.
(8) Sinclair, J.L.; Alexander, M. (1984) Role of resistance
to starvation in bacterial survival in sewage and lake water. Appl.
Environ. Microbiol. 48:410-415.
(9) Young, S.Y. (1975) Pre- and post-treatment assessment of
virus levels. Selected papers from EPA-^USDA Working Symposium.
M. Summers, R. Engler, L. Falcon, and P. Vail (eds.) American
Society of Microbiology.
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Subdivision M
155A-12 Tests "to determine expression in a marine or estuarine
environment.
(a) When required.
(1) Data on the expression of a microbial pest control agent
in a marine or estuarine environment are required by 40 CFR 158.740
to support the registration of each end-use product intended for
outdoor application on land or in fresh water and each manufacturing-
use product that legally may be used to formulate such an end-use
product when toxic or pathogenic effects are observed in any of the
following Tier I tests:
(i) Estuarine and marine animal toxicity and pathogenicity test
(154A-21); or
(ii) Plant studies - estuarine or marine (154A-22).
(2) Data on the expression of a microbial pest control agent
in a marine or estuarine environment are required by 40 CFR 158.740
to support the registration of each end-use product intended for
outdoor application in marine or estuarine environments and each
manufacturing-use product that legally may be used to formulate
such an end-use product when toxic or pathogenic effects are observed
in any of the following Tier I tests:
(i) Avian oral pathogenicity/toxicity test: Tier I. ( 154A-16) ;
(ii) Avian respiratory pathogenicity test ( 154A-17) ;
(iii) Estuarine and marine animal toxicity and pathogenicity test
(154A-21); or
(iv) Plant studies - estuarine or marine ( 154A-22).
(3) See 40 CER 158.50 and 158.740 to determine whether these
data must be submitted.
(b) Test standards.
(1) Method.
(i) Tests shall be conducted in a simulated marine or estuarine
environment (e.g., aquarium with bottom sediment) to determine
whether the MPCA is able to survive, persist, and/or
replicate in a marine or estuarine environment consisting of seawater
or brackish water and bottom sediment representative of the proposed
use site. The following parameters should be varied to determine
their effect on the survival and growth of the MPCA
population:
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Pesticide Assessment Guidelines
(A) Temperature;
(B) pH;
(C) Nutrients;
(D) Salinity;
(E) Sunlight;
(F) Oxygen content; and
(G) Turbulence.
(ii) The values selected for each parameter listed in
paragraph (b) (1) (A) through (G) of this section should be selected
to approximate the conditions expected at the intended use site.
(iii) Specialized laboratory studies designed to determine
the MPCA's growth requirements (e.g., temperature, pH, sunlight,
and oxygen) nay supplement the study described in paragraph (b) (1) (i)
of this section. A specialized lab study(ies) may demonstrate that
the MPCA will be unable to survive and persist in a marine or estua-
rine environment. In such instances, the Agency will consider this
study (ies) on an individual basis to fulfill the intent of the
testing in 155A-12 in lieu of the study described in paragraph
(b) (1) (i) of this section.
(2) Test substance. A typical end-use product or the technical
grade of the active ingredient shall be tested.
(3) Test duration. Data to establish a population decline
curve should be collected at intervals until two half-life determi-
nations have been made or until data establish that the microbial
agent population is able to maintain itself in a marine or estuarine
environment at or above the level present immediately after test
initiation.
(c) Reporting and evaluation of data. The reporting and
evaluation provisions are the same as those set forth in 155A-2.
In addition, the following information should be reported:
(1) Any changes in morphology of the microorganism in response
to changes in salinity.
(d) Tier progression. If results of this study and use
pattern information indicate that the MPCA is likely to
enter and is able to persist in a marine or estuarine environment
such that the susceptible nontarget organism(s) tested in Tier I
is likely to be exposed, then the appropriate testing in Tier III
(154A-25 through -31) is required as specified by 40 CFR 158.740.
(e) References. Refer to 154A-ll(e).
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Subdivision M
Series 156A: PRODUCT PERFORMANCE GUIDELINES FOR MICROBIAL FEST
CONTROL AGENTS
156A-1 Overview.
Submission of product performance data has been generally
waived for most products. It should be noted, however, that the
Administrator may require, on a case-by-case basis, product per-
formance data on any specific product whenever he deems that such
data are necessary to make proper benefit evaluations for decisions
whenever significant risk concerns are identified.
Certain product performance related data on MPCAs have been
solicited in these guidelines. These data include: the available
information on host spectrum and the time and the minimum effective
dosage required for a product to achieve the desired level of pest
control or other product performance standard. Such information
would ordinarily be developed (and reported) in connection with
efficacy studies and is important in the evaluation of nontarget
organism safety data.
156A—2 General provisions.
(a) Waiver of data requirements: background and policy. An
amendment to FIFRA section 3 (c) (5) provides that the Administrator
may waive data requirements pertaining to efficacy. This amendment
states:
In considering an application for the registration of a
pesticide, the Administrator may waive data requirements
pertaining to efficacy, in which event the Administrator
may register the pesticide without determining that the
pesticide composition is such as to warrant proposed
claims of efficacy.
The Agency, in testimony before Congress, stated that it is most
concerned about ensuring a product's effectiveness when a lack of
efficacy could result in adverse human health effects. In keeping
with this concern, the Administrator has deemed that all applications
for products not having a direct impact on public health may have
their efficacy requirements waived. The Agency is limiting its direct
concern to, and requiring efficacy data for, products having health-
related use patterns and products proposing new and added uses of
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Pesticide Assessment Guidelines
chemicals which have been identified as posing a risk of unreasonable
adverse effects.
(1) Product performance data will generally only be required
by 40 CER 158.160 for products of the following types:
(i) Uses of antimicrobial agents intended to control pest micro-
organisms (except bacteria, pathogenic fungi/ or viruses living on
or in man or other animals) that pose a threat to human health and
whose presence cannot readily be observed by the user, including,
but not limited to, microorganisms infectious to man in any area of
the inanimate environment;
(ii) Uses of fungicides intended for control of organisms
that produce mycotoxins.
(iii) Uses of vertebrate control agents intended to control:
(A) Commensal rat and mouse products (Norway, roof and
Polynesian rat, and house mouse);
(B) Products used to control or disperse birds from
buildings, roosts and other areas where they present health hazards;
(C) Products used to control rabies vectors such as bats,
skunks, raccoons, and canids; and
(D) Products used to control rodents considered to be plague
vectors (ground and tree squirrel, rat, and mouse).
(2) With regard to certain pesticides under suspension or
concellation action or subject to a Notice of Special Review under 40
CER 154.7, the Agency has determined that product performance data
are required to be submitted to support any new or additional uses
of products that are:
(i) Already under suspension or cancellation action be the
Agency for reason that some uses cause human or environmental
safety hazards, provided that the risks identified in the
suspension or cancellation action also apply to the new uses; or
(ii) Already subject to a Notice of Special Review, provided
that the risks identified in the Notice also apply to the new or
additional use(s).
(3) For those products for which the Administrator will
ordinarily waive the requirement for submittal of efficacy and
comparative product performance test data as indicated intends that
the registrant will generate and maintain in their records data
which is the basis for their label claims.
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186
Subdivision M
(i) With regard to conditions where the Agency may request
efficacy data but not be limited to these causes for any product,
registered or proposed for registration such as when:
(A) A lack of efficacy has been reported for it; or
(B) The Agency needs such data to evaluate benefits of the
pesticide (or of alternative pesticides) when substantial risks
have been identified; or
(C) Factors exist that make submission of such data necessary
or desirable to support the presumption that it is efficacious.
(4) Data on phytotoxicity to the target site, i.e., crops or
other desirable plants, are considered part of an efficacy evaluation
and submission is thus waived but may be called in on a case by case
basis. On the other hand, data on phytotoxicity to crops or other
plants that are nontarqet sites are considered to be data for hazard
evaluation and must be submitted on a case-by case basis as prescribed
in 154A-4 and -22. Data on the effects of MPCAs on nontarget
plants must be submitted for all such products as described in
154-22.
156A-3 Specific provisions.
(a) The following provisions apply to all microbial pest
control agents regardless of whether product performance data sub-
missions are not waived in accordance with 156A-2 (a):
(1) The available information on host spectrum shall be
reported;
(2) The tine required to achieve the desired level of pest
control or other product performance standard shall be reported;
and
(3) The minimum effective dosage (MED) necessary to achieve the
desired level of pest control or other product performance standard
shall be reported. The registrant is referred to Subdivision G,
Product Performance, for specific guidance and information on data
and reporting requirements.
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Pesticide Assessment Guidelines
Series 157A: EXPERIMENTAL USE PERMIT GUIDELINES FOR MICROBIAL PEST
CONTROL AGENTS
157A-1 Overview.
Data to support applications for Experimental Use Permits (EUPs)
for microbial pesticides generally include those data that would
ordinarily be generated during the initial stages of product deve-
lopment. For example, most product analysis information would be
developed early in the product development stages, and the Tier I
toxicology and nontarget organism toxicity tests would usually be
conducted first in preparation for registration. Unless these test
results indicate toxic, pathogenic, or other harmful properties, no
data on residues or environmental fate would ordinarily be necessary.
Product performance data requirements follow the pattern already
described in the Subdivision I Guidelines, in which waives data
for most products not dealing with public health areas are not re-
quired. However, 157A-4(g) also proposes the submittal of data on
host spectrum, and time and minimum effective dosage required to
achieve the product performance standard.
As indicated in 150A-l(c), "Data requirements for field testing",
the Agency recognizes the need to limit testing expenses in the de-
velopment of microbial pesticides and will make every effort to grant
data waiver requests where justified. Anyone planning to submit an
EUP should consult with Agency scientists in order to develop an
appropriate testing scheme.
In accordance with 40 CFR 172.3, no experimental use permit will
be required for non-food-use tests conducted on a cumulative total
of not more than 10 acres of land or not more than 1 surface acre
of water providing that the water is not used for irrigation, drinking
water, or body contact recreational activities, or that the water not
contain or affect any fish snellfish or other plants or animals used
for food or feed. The Agency is in the process of developing an amend-
ment to 40 CFR 172 to require notification prior to conducting small
scale field tests of certain genetically altered MPCAs. In the mean-
time, the Agency is following the policy published in the June 26,
1986 FEDERAL REGISTER (51 FR 23302). Under this policy, a written
notification is to be submitted to the Agency prior to canducting
small scale field tests of genetically altered or nonindigenous MPCAs.
The Agency has 90 days to determine if an EUP application is required
for this field test.
157A-2 Scope and intent.
This section series deals with the data necessary to support
the application for an Experimental Use Permit for microbial pest
control agents. These guidelines are based on FIFRA section 5 and
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188
Subdivision M
40 CFR Part 172, and they closely match the guidelines in Subdivision
I in many respects. For further information on scope and intent,
refer to 110-1 of Subdivision I.
157-3 General Provisions.
In developing plans and information for an Experimental Use
Permit application, the applicant should carefully review section
series 110 and 111 of Subdivision I. With the exception of several
cross-references to specific data in other subdivisions of the
guidelines, the provisions of those sections of Subdivision I apply
to MPCAs as well as to conventional pesticides.
When requesting preliminary assistance from Agency scientists in
determining a data testing scheme, as much of the following infor-
mation on the MPCA as possible should be available. This kind of
information will be used to determine the specific tests needed or
to determine the appropriateness of approving test waiver requests.
(1) The identity of the MPCA including;
(i) Characteristics.
(ii) Means and limit of detection.
(2) Description of its natural habitat including information
on:
(i) Predators.
(ii) Parasites.
(iii) Competitors.
(3) Information on the host range, with an assessment of
infectivity and pathogenicity to nontarget organisms.
(4) Information on the population dynamics of the
microorganism in the environment.
(5) A description of the proposed testing program including:
(i) The purpose or objectives of the proposed testing.
(ii) Designation of the pest organism(s) involved.
(iii) The State (s) in which the proposed program will be
conducted.
(iv) The specific identity of the exact location of the test
sites (s) (including proximity to residences and human activites,
surface water, etc.)
(v) The crops, fauna, flora, geographical description of
sites, modes, dosage rates, frequency, and situation of application
on or in which the pesticide is to be used.
(vi) The amount of pesticide product proposed for use.
(vii) The method of application.
(viii) A comparison of the natural habitat of the microorganism
with the proposed test site.
(ix) The number of acres, structural sites, or animals/plants
by State, to be treated or included in the area of experimental use.
(x) Procedures to be used to protect the test area from
intrusion by unauthorized individuals.
(xi) The proposed dates or period (s) during which the testing
program is to be conducted, and the manner in which superviaion of
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189
Pesticide Assessment Guidelines
the program will be accomplished.
(xii) Description of procedures for monitoring the microor-
ganism within and adjacent to the test site during the test.
(xiii) The method of disposal or sanitation of plants, animals,
soils, etc., that were exposed during and after the field test.
(xiv) Means of evaluating potential adverse effects and
methods of controlling the microorganism if detected beyond the
test area.
(6) A statement of composition for the formulation to be
tested, giving:
(i) The name and percentage by weight of each ingredient,
active and inert.
(ii) Production methods.
(iii) Extraneous microorganisms present as contaminants.
(iv) Amount and potency of any toxin present.
(v) The number of viable microorganisms per unit weight or
volume of the product (or other appropriate system for designating
the quantity of active ingredient).
The following information applies to genetically altered MPCAs:
(7) Description of the methods used to genetically alter the
microorganism.
(8) The identity and location of the rearranged or inserted/
deleted gene segment(s) in question (host source, nature, base
sequence data or restriction enzyme map of the gene(s).
(9) Information on the control region of the gene(s), and a
description of the new trait (s) or characteristic (s) that are
expressed.
(10) Data on the potential for genetic transfer and exchange
with other organisms and on genetic stability of any inserted
sequence.
(11) Data on the relative environmental competitiveness
compared to the parental strains.
157A-4 Specific data requirements.
(a) General.
(1) The following types of data are required by 40 CFR 158.740
to support an application for an Experimental Use Permit for a
MPCA:
(i) Product analysis; refer to paragraph (b) of this section;
(ii) Residues; refer to paragraph (c) of this section;
(iii) Toxicology; refer to paragraph (d) of this section;
(iv) Nontarget organisms; refer to paragraph (e) of this section;
(v) Environmental fate; refer to paragraph (f) of this section; and
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190
Subdivision M
(vi) Product performance; refer to paragraph (g) of this section.
(2) General policies related to data necessary to support an
Experimental Use Permit are delineated in Subdivision I, 112-1.
(b) Product analysis data. To support an application for an
Experimental Use Permit, the data outlined in 151A-20 through -26
apply to microbial pest control agents.
(c) Residue data. For microbial pest control agents used on
food or feed crop or whose use is expected to result in residues in
or on food or feed, no data are required unless Tier I toxicology
studies conducted under section 152A of this subdivision indicate a
potential for human hazard. Residue data developed in accordance
with section 153A of this subdivision would then be required to
obtain a temporary tolerance.
(d) Toxicology data. The following data are required by
40 CFR 158.740 to support an application for an Experimental Use
Permit:
(1) For an MPCAs:
(i) Acute oral toxicity/pathogenicity (152A-10);
(ii) Acute dermal toxicity/pathogenicity (152A-11);
(iii) Acute pulmonary toxicity/pathogenicity (152A-12);
(iv) Acute intravenous, toxicity/pathogenicity (152A-13); and
(v) Primary eye irritation (152A-14).
(vi) Hypersensitivity incidences (152A-15).
(2) For viruses used as MPCAs on food crops:
(i) All studies listed in paragraph (d) (3) of this section;
(ii) Tissue culture (152A-16).
(e) Nontaraet organism data. To support an application for
an Experimental Use Permit, nontarget organism data developed in
Tier I studies of microbial pest control agents as described in
section series 154A are required as specified in 40 CFR 158.740.
(f) Environmental fate and expression data. To support an
application for an Experimental Use Permit, data from environmental
fate and expression studies according to section series 155A are
required by 40 CFR 158.740 for those microbial pest control agents
whose Tier I nontarget organism test results (from section series
154A) indicate that Tier II studies for environmental fate and
expression should be conducted. For those pest control agents
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191
Pesticide Assessment Guidelines
whose Tier I nontarget organism test results indicate no Tier II
studies are necessary, no environmental fate and expression data are
required for the application of a Permit. In those instances where
field data from Tier II studies are required in section series 155A
for a Permit, field data from contained simulated environments or
field data conducted on similar microorganisms may suffice in
lieu of extensive field data; this policy is needed to preclude
development of extensive field data without a permit in order to
obtain information necessary to get a Permit.
(g) Product performance data.
(1) General. In general, product performance data will not
be required by 40 CER 158.740 to support the issuance of an Experi-
mental Use Permit.
(2) Exceptions.
(i) Initial permits. Efficacy data may be required, on a
case-by-case, for the following use categories:
(A) Public health uses dealing with pest microorganisms,
and vertebrate control agents; and
(B) Use of cancelled or suspended pesticides.
(ii) Extensions, renewals, and amendments. Summaries of product
performance data collected under an Experimental Use Permit may be
requested on a case-by-case basis by the Agency for purposes of
making:
(A) Determinations as to the need for additional quantities of
product requested by the applicant;
(B) Evaluations of requests for Permit extensions; and
(C) Assessments of requests for Permit renewals.
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192
Subdivision M
Series 158A: LABEL DEVELOPMENT
158A-1 Product label requirements.
Microbial pest control agents are generally subject to all
applicable labeling provisions described in Subdivision H - Labeling
Requirements for Pesticides and Devices. Labeling for microbial
agents differs principally with respect to the ingredient statement.
Some instruction regarding ingredient statements for MPCAs
can be derived from 152A-1 through -17 (Product Analysis) of this
subdivision. Also, see 156A-3 regarding label claims, directions,
precautions, and restrictions in relation to use pattern
-------
APPENDIX
S 158.170 Microbial pesticides - Product analysis data requirements.
(1) Microbial pesticides product analysis data requirements
Kind of
Data
Required
Notes
Terrestrial
Pood Non
Crop Food
GENERAL USE PATTERNS
TEST SUBSTANCE
Aquatic
Food Non-
Crop Food
Greenhouse
Food Non
Crop Food
Domestic
Outdoor
Indoor
Data to Data to Guidelines
Support Support Reference
HP EP Numbers
Product Identity [R) [RJ [R] [R]
Manufacturing Process (i) [R] pi) [R] (R]
Discussion of
Formation of
Unintentional (ii) [R] (RJ [R] [RJ
Ingredients
Analysis of Sanples (iii) [CRJ [CR]
Certification of Limits [R] R
Physical and Chemical (R] [R]
Properties
Submittal of Sanples (iv) [CR] [CR]
[R] [R]
[R] IR]
[R] [R]
ICR] ICR] [CR] [CR]
[R]
[R]
R
[R]
[R] R
[R] [R]
[CR] [CR] [CRJ [CR]
[R] [R] [R] HP EP* 151A-10
DO [R] IR) HP S TGAI EP* & 151A-11
TGAI
[R] IR] IR] HP i TGAI EP* & 151A-12
TCAI
[CR] [CR] [CRJ HP & TGAI EP* t 151A-13
TGAI
R R R HP EP* 151A-15
[R] [RJ [R] HP i EP* S 151A-16
TGAI TGAI
[CR] [CR] [CR] HP t TGAI EP*, 151A-17
PAI TGAI &
PAI
KEY: R = Required; CR = Conditionally Required; HP = Manufacturing-use product; EP* = End-use product, (asterisk identifies
those data requirements that end-use applicants (i.e., "f emulators") must satisfy, provided that their active
ingredient(s) is (are) purchased from a registered source); TGAI * Technical Grade of the Active Ingredient;
[] = Brackets (i.e., [R], [CR] indicate data requirements that apply when an experimental use permit is being sought.
(i) If an experimental use permit is being sought, a schematic diagram and/or description of the manufacturing process
will suffice if the pesticide is not already under full scale production.
(ii) If the product is not already under full scale production and an experimental use permit is being sought, a
discussion of unintentional ingredients shall be submitted to the extent this information is available.
(iii) Required to support registration of each manufacturing-use product and end use products produced by an
integrated formulation system. Data on other end use products will be required on a case-ty-case basis. For pesticide in
the production stage, a rudimentary product analytical method and data will suffice to support an experimental use permit.
(iv) Routinely required for products produced by an integrated formulation system. Required on a case-ty-case basis
for other products or materials.
(2) Hicrobial pesticides residue data requirements
GENERAL USE PATTERNS
TEST SUBSTANCE
Kind of
Data Required
Residue Data
Notes
(i)
Terrestrial
Food Non-
Crop Food
ICR]
ICR]
Aquatic
Food Non-
Crop Food
[CRJ ICR]
Greenhouse
Food
Crop
[CR]
Non-
Food
[CR]
Forestry
[CRJ
Domestic
Outdoor
[CRJ
Indoor
[CRJ
Data to
Support
HP
-
Data to
Support
EP
-
Guidelines
Reference Number
153A
Key: CR » Conditionally Required Data; End-use product; HP « Hanufacturing-use product; [] « Brackets
(i.e., [CR]) indicate data requirements that apply when an experimental use permit is being sought.
(i) Residue data requirements shall apply to microbial pesticides when Tier II or Tier III
toxicology data are required.
-------
(3) Hicrobial pesticides toxicology data requirements.
GENERAL USE PATTERNS
Terrestrial Aquatic Greenhouse
Food Non Food Non- Food Non- Domestic Indoor
Notes Crop Food Crop Food Crop Food Forestry Outdoor Use
TEST SUBSTANCE
Data to
Support
HP and EP
Guideline
Reference
Numbers
Tier I
Acute oral tox/fcath
Acute dermal tox
Acute pulmonary tox/path
[R] [R] [R] [R] IR) IR1
IR] [R]
IR]
[R]
[RJ
[R]
R
[Rl
[R)
TCAI
HP & EP
TGAI
152A-10
152A-11
152A-12
Acute intravenous tox/path (i)
Primary eye irrit.
Hype rsens itivity
incidents
Cell culture
Tier II
Acute tox
Subchronic tox/patl
(ii)
(iii)
(iv)
(v)
h (vij(vii)
IR1
tR)
R
IR]
CR
CR
[R]
(R)
R
[R]
CR
CR
[R]
[R]
R
[R]
CR
CR
[R]
[Rl
R
[R]
CR
CR
[R]
[R]
R
[R)
CR
CR
[R]
[R]
R
[R]
CR
CR
IR)
[R]
R
[R]
CR
CR
[R]
[R]
R
IR]
CR
CR
[R] TGAI
IR] HP 6 EP
R
tR] MIF
CR
CR
152A-13
152A-14
152A-15
152A-16
152A-20
152A-21
Tier III
Reproductive/fertility
effects (viii)
Oncogenicity (ix)
Immunodeficiency (x)
Prjmate infectivi^
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
152A-30
152A-31
152A-32
152A-33
KE¥; R = Required; CR = Conditionally Required; MIF = Most Infective Form; HP = Manufacturing-use Product; EP « End-use
Product; TGAI = Technical Grade of the Active Ingredient; [] = Brackets (i.e., (R), [CRJ) indicate data requirements
that apply when an experimental use permit is being sought.
(a) If an MP is different than the TGAI, then an acute oral toxicity study (81-1) and an acute inhalation study
(81-3) as required in 158.340 for chemical pesticides is to be done with the MP as the test substance. If the EP is
different than the MP and the TGAI, then an acute oral toxicity study (81-1) and an acute inhalation study (81-3) as
required in 158.340 for chemical pesticides is to be done with the EP as the test substance. End-use applicants (i.e.,
•fotnulators") trust satisfy these data requirements for the end-use products provided that their active ingredient(s)
is (are) purchased from a registered source.
(b) Notes.
(i) Data not required for products whose active ingredient is a virus.
(ii) Data not required if it is demonstrated (usually by product label statement inclusion)
that workers/applicators eyes will be appropriately protected during times of exposure to aerosols
of the MPCA.
(iii) Hypersensitivity incidents oust be reported, if they occur.
(iv) Data required for products whose active ingredient is a virus.
(v) Data required when toxicity, in the absence of pathogenicity and significant infectivity is
observed in Acute oral, denial, and pulmonary studies (Tier I). Rcute(s) of exposure correspond to routes
where toxicity was observed in Tier I studies.
(vi) Data required when significant infectivity and/or unusual persistence is observed in the absence of
pathogenicity or toxicity in Tier I studies. Routes of exposure (oral and/or pulmonary) correspond to routes
in Tier I studies.
(vii) Also nay be required to evaluate adverse effects due to microbial contaminants or to toxic by-products.
(viii) Data required when any of the following criteria are met:
1) Significant infectivity of the MPCA was observed in test animals in the Tier II subchronic study (152A-22)
and in which no significant signs of toxicity or pathogenicity were observed.
2) The MPCA is a virus which can persist or replicate in mammalian cell culture lines (152A-19).
3) The MPCA is not amenable to thorough taxonomic classification, and is related to organisms known to be
be parasitic for mammalian cells.
-------
4) The MPCA preparation is not sufficiently well purified, but it is indicated that the preparations
nay contains contaminants which are parasitic for mammals.
(ix) Data may be required for products known to contain, or suspected to contain, oncogenic viruses.
(x) Data may be required for products known to contain or suspected to contain, viruses that can interact
in an adverse manner with components of mammalian imnune systems.
(xi) Data may be required for products that are known to contain, or are suspected of containing intracellular
parasites of mammalian cells, or for products that exhibit pathogenic characteristics in Tier 1 or for known human
pathogens that have been 'disarmed*.
(4) Hicrobial pesticides non-target organism and environmental expression data requirements.
GENERAL USE PATTERNS
Tier I
Avian oral
Avian inhalation test
Wild mammal testing
Freshwater fish
testing
Freshwater aquatic
invertebrate testing
Estuarine and marine
animal testing
Nontarget plant studies
Nontarget insect
testing
Honey bee testing
Tier II
testinji
Freshwater environmental
expression tests
Marine or/estuarine
environmental expression
tests
Terrestrial
Food Non
Notes
(iv) 'X *
(v)
(Ml)
(i)
(vi)
al (vii)
1 (viii)
(ix,x)
Crop
[RJ
CR
CR
[R]
[R]
CR
[RJ
[R]
[RJ
CR
CR
CR
Food
CR
CR
[R]
[R]
CR
[RJ
[RJ
[RJ
CR
CR
CR
Aquatic
Food Non-
Crop
[RJ
CR
CR
[R]
[R]
CR
[RJ
[R]
[RJ
CR
CR
CR
Food
[RJ
CR
CR
[R]
[R]
CR
[RJ
[R]
[RJ
CR
CR
CR
Greenhouse
Food Non
Crop Food Forestry
CR CR [RJ
CR CR CR
CR
CR CR [R]
CR CR [R]
CR
[RJ
CR CR [R]
CR CR (RJ
CR
CR
- CR
Domestic
Outdoor
[RJ
CR
CR
CR
CR
CR
[RJ
[RJ
[RJ
CR
CR
CR
Indoor
Use
CR
CR
—
CR
CR
-
CR
™
-
-
-
TEST SUBSTANCE
Data to
Support
MP
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI orTEP
TUAI orTEP
TGAI orTEP
Data to
Support
EP
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
TGAI
Guidelines
Reference
Numbers
154A-16
154A-17
154A-18
154A-19
154A-20
154A-21
154A-22
154A-23
154A-24
TGAI orTEP 15SV-18
TGAI orTKP 155A-19
TGAI orTEP 155A-20
Tier III
Terrestrial Wildlife &
Aquatic (xi) CR
Organism Testing
Avian Chronic pathogenicity(xii) CR
and reproduction test
Aquatic invertebrate (xiii) CR
range testing
Pish Life Cycle Studies (xiii) CR
Aquatic Ecosystem Test (xv) CR
Special Aquatic Tests (Reserved)
Nontarget Plant Studies (xvi) CR
Tier IV
Simulated and Actual
Field Tests (Birds,
(Mammals)
Simulated and Actual
Field Tests (Aquatic
Organisms)
Simulated and Actual
Field Tests (insect
Predators, Parasites)
Simulated and Actual
Field Tests (Insect
Pollinators)
(xvii)
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
(xiv)
(xviii,
xix)
(xviii,
xix)
(xviii,
xix)
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
CR
TGAI orTEP
TUAI
TGAI
TGAI
TGAI
TGAI
TGAI orTEP
TGAI
TGAI
TGAI
TGAI
TEP
154A-25
154A-26
154A-27
154A-28
154A-29
154A-30
154A-31
TEP
TEP
TEP
TEP
TEP
TEP
TEP
TEP
154A-33
154A-34
154A-3S
154A-36
KEY; R = Required
CR « Conditionally Required
[] » Brackets {i.e., [Rl, [CR]) indicates data requirements that apply to products for which an experimental use
permit is being sought.
HP = Manufacturing-use Product
TEP » Typical End-Use product
TGAI » Technical grade of the active ingredient.
EP « End-use product, PAI = "Pure" active ingredient.
-------
Notes.
(i) Tests for pesticides intended solely for indoor application will be required on a case-by-case besis,
depending on use pattern, production volume, and other pertinent factors.
(ii) Preferable test species are: bobwhite quail or mallard for avian acute oral and inhalation studies;
rainbow trout for freshwater fish studies.
(iii) An injection route of exposure may be used in lieu of the oral route in some cases for the avian
acute study.
(iv) Data required when the nature of the MPCA and/or its toxins indicates potential pathogenicity to birds.
(v) Required on a case-by-case basis if results of tests required by paragraph (c)(l) of this section are
inadequate or inappropriate for assessment of hazards to wild animals.
(vi) Required when prodjct is intended for direct application into the estuarine or marine environment or
expected to enter this environment in significant concentrations because of expected use or mobility pattern.
(vii) Required when toxic or pathogenic effects are observed in any of the following Tier I tests for microbial
pest control agents:
(A) Avian acute dose oral or avian inhalation tests.
(B) Wild normals toxicity and pathogenicity test.
(C) Plant studies - terrestrial.
(D) Honey bee toxicity/pathogenicity test.
(E) Testing for toxicity/pathogenicity to insect predators and parasites.
(viii) Required when toxic or pathogenic effects are observed in any of the following Tver I test for microbial
pest control agents:
(A) Freshwater fish toxicity and pathogenicity testing.
(B) Freshwater aquatic invertebrate toxicity and pathogenicity test.
(C) Plant studies aquatic.
(ix) Required if product is applied on land or in £ resh water and toxic or pathogenic effects are observed
in any of the following Tier I tests for microbial pest control agents:
(A) Estuarine and marine animal toxicity and paUJpgenicity test.
(B) Plant studies - estuarine or marine.
(x) Required if product is applied in marine or estuarine environments and toxic or pathogenic effects are
observed in any of the following Tier I tests:
(A) Avian acute oral test.
(B) Avian inhalation test.
(C) Estuarine and marine animal toxicity and pathogenicity test.
(xi) Required when toxic effects on nontarget terrestrial wildlife or aquatic organisms are reported in one
or more Tier I tests and results of Tier II tests indicate exposure of the microbial agent to the affected nontarget
terrestial wildlife or aquatic organisms.
(xii) Requited when:
(A) Pathogenic effects are observed in Tier 1 avian tests.
(B) Chronic, carcinogenic, or teratogenic effects are reported in tests required in (c)(l) of this section
for evaluating hazard to humans and domestic animals.
(C) Tier II Environmental expression testing indicates that exposure of terrestrial animals to the microbial
agent is likely.
(xiii) Required when product is intended for use in water or expected to be transported to water from the
intended use site, and when pathogenicity or infectivity was observed in Tier I aquatic tests.
(xiv) Required when both of the following conditions are met:
(A) Pathogenic effects at actual or expected field residue exposure levels are reported in Tier III.
(B) The Agency determines that quarantine methods will prevent the microbial pest control agent from
contaminating areas adjacent to the test area.
(xv) Required if, after an analysis of the microbial agent's properties, the individual use patterns, and
the results of previous nontarget organism and environmental expression tests, it is determined that use of the
microbial agent may result in adverse effects on the nontarget organisms in aquatic environments, including
those of the water column and bottom sediments. When a microbial pest control apent is used in or is expected
to transport to water from the intended use site, major considerations for requiring these infectivity tests
include, but are not limited to:
(A) Infectivity or pathogenicity demonstrated in previous testing.
(B) Viability of the microorganism in natural waters as demonstrated in Tier II tests.
(xvi) Required if the product is transported from the site of application by air, soil, or water or
transmission by other animals, and nontarget plant pathogenicity is observed in Tier I tests. The exent of
movement will be determined by the environmental expression tests in Tier II.
(xvii) The Agency expects that Tier IV requirements would be imposed retrospectively - after product
registration as post registration monitoring, since it is unlikely a registrant would pursue registration of a
microbial agent posing potential hazards such that testing beyond Tier III is required.
(xviii) Short term simulated or actual field studies are required when it is determined that the product is
likely to cause sdv-rs^ short-term or acute effects, based on consideration of available laboratory data, use
patterns, and exposure rates.
(xix) Data from a long-term simulated field test (e.g., where reproduction and growth of confined populations
are observed) and/or an actual field test (•>. -j., where reproduction and growth of natural populations are observed)
are required if laboratory data indicate adverse long-term, cumulative, or life-cycle effects may result from
intended use.
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