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Pesticide Assessment
Guidelines
Subdivision M
Biorational Pesticides
Printed on Recycled Paper
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EPA
November, 1982
PESTICIDE ASSESSMENT GUIDELINES
Subdivision M
Biorational Pesticides
Prepared by
F. S. Betz
W. R. Beusch
E. B. Brittin
R. Carsel
S. Z. Cohen
R. W. Hoist
A. Keller
I. N. Mau«r
W. Roeasler (retired)
D. Urban
A. Vaughan
W. Woodrow
Guideline Coordinator
Robert K. Hitch
Hazard Evaluation Division
Office of Pesticide Programs
0-S. Environmental Protection Agency
Office of Pesticide and Toxic Substances
Washington, D.C. 20460
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EPA-540/9-82-028
October 1982
Pesticide Assessment Guidelines
Subdivision M
Biorational Pesticides
Prepared by
F.S. Betz
W.R. Beusch
E.B. Brittin
R. Carsel
S.Z. Cohen
R.W. Hoist
A. Keller
I.N. Mauer
W. Roessler (retired)
D. Urban
A. Vaughan
W. Woodrow
Guidelines Coordinator
Robert K. Hitch
Hazard Evaluation Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
Office of Pesticides and Toxic Substances
Washington, O.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 (FXFRA). Protocols are provided for
determining the chemical fate of these pesticides in the environment
and for evaluating their potential adverse effects on humans and
other nontarget organisms. Subdivision M is a nonregulatory com-
panion to 40 CFR Part 158, Data Requirements for Registration. Zt
has been the subject of comment at a series of public meetings,
the last of which occurred in July 1982. Data requirements estab-
lished by 40 CFR Part 158 are discussed in Subdivision M so that
it can be read as a complete package and so that the testing pro-
cedures for biochemical and microbial pesticides can be explained
in their proper context.
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SUBDIVISION M: GUIDELINES FOR TESTING BIORATIONAL PESTICIDES
Table of Content*
DISCUSSION
I. Scop* of Subdivision M
II. Background of Subdivision M
III. Organization and Philosophy of Subdivision M
IV. Product Analysis
v. Toxicology
VI. Residue Analysis
VII. Nontarget Organism Hazard
VIII. Environmental Fate and Expression
IX. Product Performance
X. Experimental Use Permits
XI. Promoting Blorational Pesticides By
Modifying Label Claims
GUIDELINES
Series 150:
150-1
150-2
150-3
150-4
Series 151:
151-1
151-2
Subscries: 151A:
151-10
151-11
151-12
151-13
151-14
151-15
151-16
Overview, Definitions, and General
Provisions
Overview
Definitions
General provisions
Reporting of data
Product Analysis Guidelines
General Information
Through 151-9 [Reserved]
Product Analysis Guidelines for
Biochemical Pest Control Agents
Product identity and disclosure of
ingredients
Manufacturing process
Discussion on the -formation of
unintentional ingredients
Analysis of samples
[Reserved]
Certification of ingredient limits
Analytical methods for certified limits
Page
1
5
6
9
10
31
33
77
85
86
86
89
89
93
96
100
100
102
102
103
103
103
-------
Table of Contents
(Continued)
151-17
151-18
Subseries 151B:
151-20
151-22
151-23
151-24
151-25
151-26
151-27
Physical and chemical properties
Subnittal of samples
[Reserved]
Product Analysis Guideline*
Product identity and disclosure of
ingredients
Manufacturing process
Discussion on formation of unintentional
ingredients
Analysis of samples
[Reserved]
Certification of ingredient limits
Physical and chemical properties
Submit tal of samples
Page
104
104
Series 152: Toxicology Guidelines
152-1 General information
152-2 Through 1S1-9 [Reserved)
Subseries 152A: Toxicology Guidelines
Group A-1: Tier I Testing
152-10 Acute oral toxicity study
152-11 Acute dermal toxicity study
152-12 Acute iirialation toxicity study
152-13 Primary eye irritation study
152-14 Primary dermal irritation study
152-15 Hyper sensitivity study
152-16 Hypers ens itivicy incidents
152-17 Studies to detect geno toxicity
152-18 Cellular immune response studies
Group A-2: Tier XI Testing
152-19 Mammalian mutagenicity tests
152-20 Sub chronic oral dosing studies
152-21 Subchronic dermal toxicity sta
152-22 Subchronic inhalation toxicity
study
106
108
108
109
109
109
110
112
117
118
119
120
121
122
122
123
125
129
133
134
135
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152-23
152-24
152-25
Group A-3:
152-26
152-27
152-29
Scries 152B:
Group B-1:
152-30
152-31
152-32
152-33
152-34
152-35
152-36
152-37
152-38
152-39
Group B-2:
152-40
Table of Contents
(Continued)
Termtogenicity studies
Cellular immune response studies
[Reserved]
Tier III Testing
Chronic exposure study
Through 152-28 [Reserved]
Oncogenicity studies
Toxicology Guidelines for
Microbial Pest Control Agents
Tier Z Testing
Acute oral toxicity/inf activity study
with microbial pest control
Acute dermal toxicity/infactivity
study'with microbial pest control
agents
Acute inhalation toxicity/infactivity
study with microbial pest control
agents
Intravenous, intracerebral, and intra-
periboneal toxicity/infectivi ty studies
with microbial pest control agents
Primary dermal irritation study
with microbial pest control agents
Primary eye irritation study with
microbial pest control agents
Hypersensitivity study with micro-
bial pest control agents
Hypersensitivity incidents with micro-
bial pest control agents
Effects of microbial pest control agents
on the cellular immune response system
Tissue culture tests with viral agents
Tier XI Testing
Acute oral toxicity/infectivity studies
with viral or protozoan agents
Page
136
137
139
140
142
145
149
156
160
166
167
168
170
174
ill
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Table of Contents
(Continued)
152-41
152-42
152-43
152-44
152-45
152-46
152-47
152-48
152-49
Group B-3:
152-50
152-51
152-52
152-53
Series 153:
153-1
153-2
153-3
153-4
Series 154:
154-1
154-2
Subseries 154A:
Group A-1:
154-6
Page
Acute inhalation toxidty/infectivity study
with viral or protozoan agents 177
Sub chronic oral doling study with protozoa 180
Acute intraperitoneal or intracerebral
toxicity/infectivity tests with bacteria,
fungi, and protozoa 181
Primary dermal irritation study with microbial
agents 182
Primary eye irritation study with microbial
agents 183
Effects of microbial pest control agents on
the cellular immune response system 183
Teratogenieity tests with viral agents 186
Bacterial and fungal virulence enhancement 186
Mammalian mutagenicity testing with
microbial pest control agents 189
Tier XII Testing
Chronic feeding study 190
Oncogeniclty studies 190
Mutagenicity testing 191
Teratogenieity studies 192
Residue Analysis Guidelines
Overview 192
[Reserved]
Residue data requirements for
biochemical pest control agents 193
Residue data requirements for
microbial pest control agents 194
Nontarget Organism Hazard Guidelines
General Information 196
Through 154-5 [Reserved]
Nontarge t Organism Hazard Guideline*
for Biochemical Agents
Tier I Testing
Avian single dose oral toxicity test 210
iv
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Table of Concents
(Continued)
Page
154-7 Avian dietary toxicity test
154-8 Freshwater fish scute bioassay
154-9 Freshwater aquatic invertebrate acute
bioassay
154-10 Plant studies
154-11 Nontarget insect testing
Group A-2:
Tier III Testing
154-12 Terrestrial wildlife testing
154-13 Aquatic animal testing
154-14 Plant studies
154-15 Nontarget insect testing
212
213
215
217
219
220
221
221
222
Subseries 154B:
Group B-1:
154-16
154-17
154-18
154-19
154-20
154-21
154-22
154-23
154-24
Group B-2:
154-25
154-26
Nontarget Organism Hazard Guidelines
for Hicrobial Agents
Tier I Testing
Avian single-dose oral toxicity and
pathogenicity test
Avian injection pathogenicity test
Wild mammal toxicity and pathogenicity
tes ting
Freshwater fish toxicity and
pathogenicity testing
Freshwater aquatic invertebrate
toxicity and pathogenicity testing
Estaurine and marine animal toxicity
and pathogenicity tests
Plant studies
Sontarget insect testing for toxicity/
pathogenicity to insect predators and
parasites
Boney bee toxicity/pathogenicity
tests
Tier III Testing
Terrestrial wildlife and aquatic
organism toxicity testing
Long-term avian pathogenicity and
reproduction test
223
226
229
232
238
244
251
252
254
255
256
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Table of Contents
(Continued)
154-27
154-28
154-29
154-30
154-31
154-32
Group B-3:
154-33
154-34
154-35
154-36
Series 155:
Subseries 155A:
155-1
155-2
155-4
155-5
155-6
155-7
155-8
155-9
155-10
155-11
155-12
155-13
155-14
Definitive aquatic animal
pathogenic!ty tacts
Fish eabryo-larvae studies and life
cycle studies of fish and aquatic
invertebrates
Aquatic ecosys ten-pa thogenicity tests
Special aquatic tests-tissue culture,
microorganisms/stress interaction
tests [Reserved]
Plant studies
[Reserved]
260
261
264
266
Tier IV Testing
i ting
Simulated and actual field t<
for mammals and birds
Simulated or actual field testing
for aquatic organisms
Simulated or actual field testing for
Insect predators and parasites [Reserved]
Simulated or actual field testing
for insect pollinators [Reserved]
Environmental Fate and Expression
Guidelines
Tier ZZ Environmental Fate Guidelines
for Biochemical Pest Control Agents
General Information
And 155-3 [Reserved]
Volatility
Dispenser - water leaching
Absorption - desorption
Octanol/water partition coefficient
Ultraviolet - visible absorption
Hydrolysis
Aerobic soil metabolism
Aerobic aquatic metabolism
Soil photolysis
Aquatic photolysis
[Reserved]
267
269
272
276
277
279
279
280
281
281
282
283
284
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Subseries 15 SB:
155-15
155-16
155-17
155-18
155-19
155-20
155-21
Group 156:
156-1
156-23
Series 157:
157-1
157-2
157-3
157-4
Series 158:
158-1
Table of Contents
(Continued)
Tier XI Environmental Expression
Guideline* for Hicrobial Agents
General information
General test standards
Reporting and evaluation of data
Tests to determine expression in
a terrestrial environment
Tests to determine expression in a
freshwater environment
Tests to determine expression in a
marine or estaurine environment
Through 155-23 [Reserved]
Product Performance Guidelines
General Provisions
Specific Provisions
Experimental Use Permit Guidelines
Scope and intent
[Reserved]
General provisions
Specific provisions
Label Development
Product label guidelines
Page
285
288
289
290
294
297
299
300
301
301
301
304
vii
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1
I. SCOPE OP SUBDIVISION M
A. Content* of Subdivision.
Subdivision M provides guideline* for testing and information
on data submission concerning the following eight section series
and topics:
151 Product analysis
152 Toxicology
153 Residue chemistry
154 Montarget organism hazards
155 environmental fate and expression
156 Product performance
157 Experimental use permit data
158 Label development
Proposed rule, 40 CTR Part 158, specified the Jcind of data and
information that must be submitted to EPA to support the registration
of each pesticide under the Federal Insecticide, Fungicide and
Rodenticide Act. The Agency intends to promulgate Part 158 as a
final rule during 1983. This subdivision provides detailed informa-
tion relating to the data requirements listed in 40 CTR § 158.165
including the conditions under which each data requirement is appli-
cable, the standards for acceptable testing, stated with as much
specificity as the current scientific disciplines can provide, and
the information that should be included in a test report.
1. Scope of bierational pesticides. Biorational pesticides
are a distinct group, inherently different from conventional pesti-
cides. Some of the characteristics that typically distinguish
biorational from conventional pesticides are their unique non-toxic
mode of action, low use volume, target species specificity, and
natural occurrence. Based on these characteristics, the Agency
expects that many biorational pesticides pose lower potential
risks than conventional pesticides. Therefore, these pesticides
are subject to a different set of data requirements, as specified
in $158.33. Biorationals are comprised of two major categories of
pesticides: the biochemical pest control agents (e.g., pheromones,
hormones, natural insect and plant growth regulatoriMknd enzymes)
and the microbial pest control agents (e.g., microorganisms). The
relationships between conventional pesticides V*ibXbgi£«JL"8ontrol
agents, and biorational pesticides are illustrated in Figure 1.
Pesticides to be included in these categories are determined as
follows:
(2) The chemical must be naturally occurring, or if the chem-
ical is synthesized by man, then it must be structurally identical
to a naturally occurring chemical. For a synthetic chemical to be
identical in chemical structure to a naturally occurring chemical,
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the molecular structure(s) of the major component (s) of the syn-
thetic chemicalt») must be the same as the molecular structures(*)
of the naturally occurring analogue(s). Minor differences between
the rtereochemical isomer ratios (found in the naturally occurring
compound compared to the synthetic compound) will normally not rule
out a chemical being classified as a biorational unless an isomer
is found to have significantly different toxlcological properties
than another isomer.
There are situations where a candidate chemical possesses many
characteristics of a biorational pesticide, but does not technically
meet the two criteria established for defining biochemical pest con-
trol agents. The Agency will evaluate chemlcal(s) that are substant-
ially similar to biochemical! on a case-by-case basis to determine
whether the chemical should be classified as a biorational or a con-
ventional pesticide. For example, a case-by-case evaluation would be
required if the exact molecular structure of the naturally occurring
compound(s) is (are) unknown, or if the synthetic chemical is closely
related to but not identical in structure to the naturally occurring
compound, or If the mode of action is different in the target, com-
pared to non-target organism*.
Zn these case-by-case situations, the criteria the Agency will
use to determine whether the chemical(s) is (are) a biorational
pesticide(s) include: 1) the chemical and toxlcological signifi-
cance of the differences in chemical structure, 2) the mode of
action of the synthetic analogue in the target species as compared
to the mode of action of the naturally occurring compound, and
3) differences In toxicity (as demonstrated in at least the Tier I
screening tests for biorational pesticides, as specified in 40 CFR
§ 158.165) between the naturally occurring chemical and the synthetic
analogue. If a synthetic analogue is found to demonstrate direct
toxicity to any non-target organisms, based on Tier I testing, then
the analogue may or may not be classified as a biorational pesticide.
In evaluating these case-by-case situations, the Agency may find
it appropriate to classify a chemical as a biorational, yet still
impose certain conventional pesticide data requirements for some dis-
ciplines (e.g., non-target organisms and environmental fate) and the
biorational pesticide data requirements for the remaining disciplines.
Biochemical agents fall into four general biologically functional
classes: semiochemlcals, hormones, natural plant regulators, and
enzymes. They are discussed below.
Semiochemlcals. Chemicals esiitt«d..by pl*nts or animals that
modify the behavior of receptor organism* of like or different
kinds are termed semiochemlcals. They include pheromones, allomones,
and-kairomoaes. -Ph^rq«o»e»-ar*-
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modify the behavior of a different species, to the benefit of the
emitting species. Kairomones are chemicals emitted by one species
which modify the behavior of a different species to the benefit of
the receptor species.
Hormones. Hormones are biochemical agents are are synthesized
in one part of an organism and translocated to another where they
have controlling, behavioral, or regulating effect.
Natural plant regulators. Hatural plant regulators are
chemicals produced by plants that have toxic, inhibitory, stimulatory,
or other modifying effects on the saae or other species of plants.
Some of these are termed "plant hormones" or "phytohormones."
Enzynes. For the purposes of this subdivision, enzymes are
protein molecules that are the instruments for expression of gene
action and that catalyze chemical reactions.
(b) Microbial pest control agents. The biorational pesticides
referred to as microbial pest control agents include (but are not
limited to) bacteria, fungi, viruses, and protozoans. The guide*
lines apply to all microbial p«st control agents used as pesticides,
including not only those that are naturally occurring, but also
those that are strain-improved. Each variety or subspecies of a
microbial pest control agent should be tested. Data necessary to
support the registration of genetically-engineered microbial pest
control agents will be determined by the Agency on a case-by-case
basis except where specific requirements are specified in Part 158.
Pest control organisms such as insect predators, nematodes,
and macroscopic parasites are not considered biorational pesticides,
and are exempt from the requirements of FIFRA as authorized by sec.
25(b)(1) of FIFRA and specified in the Exemption from Regulation of
Certain Biological Control Agents published in the Federal Register
of June 2, 1982 (47 FR 23928).
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figure 1—RELATIONSHIPS BETWEEN CONVENTIONAL PESTICIDES, BIOLOGICAL
CONTROL AGENTS AND BIORATIONAL PESTICIDES
Pest Control Agents
Convention*! Chemical
Pesticides
I
V
Biological and Biologically
derived Pest Control Agents
Biorational Pesticides
I
V
All Other Living
Pest Control Agent*
- Insect Predators
- Macroscopic Parasites
- Nematodes
Biochemical Pest
Control Agents
- Seaiochemicals
- Hormones
- Natural Plant
Regulators
- Enzymes
I
V
Microbial Pest
Control Agents
- Bacteria
• Fungi
- Protozoa
• Viruses
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XI. BACKGROUND OF SUBDIVISION M
A* General.
EPA proposed the introduction, produce chemistry, environmental
chemistry, and fish and wildlife guidelines as Part: 163 of Title 40
in the Code of Federal Regulations published in the Federal Register
of July 10, 1978 (43 FR 29696). In the 1978 proposed guidelines/
§ 163.40-4 of the Introduction to the Guidelines (Subpart B) set
forth EPA's policy for registering biological control agents as
follows: "The data requirement* for those living organisms or
viruses which are pesticides will be determined on a case-by-case
basis after consultation with the Agency.*
EPA issued "Regulation of 'Biorational1 Pesticides; Policy
Statement and Notice of Available Background Document" published in
the Federal Register of May 14, 1979 (44 FR 23994). In this
statement of proposed policy, EPA defined biorational pesticides to
include biological pest control agents and certain naturally
occurring biochemicals which are inherently different in their mode
of action from most organic and inorganic (i.e., conventional)
pesticide compounds currently registered with EPA. The statement
also presented EPA's intent to develop, in the next 24 months,
guidelines setting forth the human health and environmental safety
data requirements for the registration of biorational pesticides.
These guidelines provide information regarding the conduct of
acceptable tests, guidance on evaluation and reporting>of data,
further guidance on when data are required, and examples of pro-
tocols. In addition, scientific publications are cited in the
guidelines to provide useful information for designing test
protocols.
B. Foraulators' Exemption.
In the Preamble to the 1978 proposed Guidelines, EPA asked for
public comment on the question of which data requirements should be
extended to manufacturing-use products. After serious consideration
of this issue, the Agency concluded that extending the data require-
ments to such pesticides is appropriate. The Agency was influenced
by the views of commenters on this issue who generally favored a data
submission requirement which makes the basic manufacturer of an
active ingredient responsible for providing most of the data.
Therefore, a section of 40 CFR Part $ 158, entitled •Formula-
tors' exemption" (§158.50) requires a registrant of a manufacturing-
use product to submit (or cite) any data pertaining to the safety
of an active ingredient in its product if the saae data are required
to support the registration of an end-use product that could legally
be produced fro* the registant'* manufacturing-use products. (An
end-use product is a pesticide product bearing label directions
for izsiediate end-use as a pesticide.) Section 158.50 also
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provide* that such data oust be submitted by an applicant for
registration of the end-use product, except that the producer of
the end-use product will generally not have to submit or cite data
pertaining to registered products which the end-use producer
purchases and uses to formulate the end-use product. This decision
reflects the Agency's expectation that manufaeturing-use product
registrants will be the major source of registration data/ and that
end-use product femulators will, in sort cases, need to supply
much less data. This decision is consistent with the provisions
of, and Congressional intent behind, sec. 3(c)(2)(0) of PXFRA,
which provides that:
No applicant for registration of a pesticide who pro-
poses to purchase a registered pesticide from another
producer in order to formulate such purchased pesticide
into an end-use product shall be required to—
(i) submit or cite*data pertaining to the safety
of such purchased product; or
(li) offer to pay reasonable compensation otherwise
required by [§ 3(c)(1)(D)) of FXFfiX] for use of any such
data.
Implicit in sec* 3(c)(2')(D) is Congress1 expectation that it
would be the registrant of the manufacturing-use product who would
provide significant amounts of data pertaining to the safety of its
product. (See, e.g., Sen. Rep. So. 334, 95th Cong., 1st Sess.,
pp. 8-9.)
Moreover, if data requirements were imposed solely on regis-
trants of end-use products, sec. 3(c)(2)(D) might be read to prevent
the Agency from obtaining data on the grounds that the data pertain
to the safety of a purchased product.
III. ORGANIZATION AND PHILOSOPHY OF SUBDIVISION M
B. Approach to Testing.
The approach taken in developing these guidelines was
significantly influenced by EPA's proposed policy (44 FR 23994)
that states:
In regulating biorational pesticides K7A will recognize
that biorational pesticides are inherently different from
conventional pesticides, and will take steps to substan-
tiate by scientific data the expectation that many classes
of biorational control agents pose lower potential risks
than conventional pesticides. Although biorational
pesticide registrants will not be relieved of the burden
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of proof of their safety, the Agency will tske into
account the fundamentally different -modes of action of
biorationals and the consequent lower rj t*r« of adverse
effects from their use.
The most important inherent differences between biorational
pesticides and conventional pesticides are: target species specifi-
city, generally nontoxic mode of action, and natural occurrence of
the biorational agents. These factors have made the use of many
biorationals practicable only under the direct supervision of a
skilled entomologist, plant pathologist, weed scientist or inte-
grated pest management (XPM) consultant-. 1PM techniques maximize
usage of natural pest controls and cultural practicesj therefore
all introduced pest control materials (biorationals as well as
conventional chemical pesticides) are used with discretion. These
factors provide the basis for the Agency's expectation that many
classes of biorational pest control agents pose a lower potential
hazard than conventional pesticides and support the approach to
testing discussed in the following paragraphs.
Three elements form the basis of XFA's approach and meet the
intent of the above policy. They are: exposure criteria (for
:als), maximam hazard testing, and a tier testing scheme.
1. "Exposure criteria. Certain factors often .associated with
biochemical pest control agents or their use, significantly limit
the agent's potential for human and other nontarget organism expo-
sure and, therefore, hazard. One or all of these factors provide a
basis for criteria for reduced data requirements. These criteria
are: low exposure pesticide formulation, low rate of application,
nonaquatic use site, and high volatility. These criteria are
described below.
(a) Low exposure pesticide formulation. Certain biochemicals
are formulated in passive dispensers such as hollow fibers, tape,
or fixed traps. The Likelihood of oral or dermal human exposure
and direct exposure of other nontarget organisms is JAW when
pesticides are formulated in this manner.
(b) Xaw rate of application. Certain biochemical pesticides
(e.g., semiochemicals) will be used in the field at very low rates.
Low use rates can be tentatively described as 0.7 ounces (20 grams)
or less active ingredient per acre per application, with rates of 1
to 5 grams per acre more common. Such low rates of application
result in squally low and possibly nonhazardons levels of exposure
to humans and other nontarget organisms, these figures are based
on label information of currently registered pheromones and on the
labels of those still under experimental use permits.
(c) Non-aquatic use site. Biochemical pest control agents
on land pose less of a risk to nontarget aquatic species
-------
8
than those Applied directly to water. Therefore, biochemical agents
applied directly to land are more likely to qualify for reduced
testing for nontarget organism hazard than those applied directly
to water.
(d) High volatility. High volatility is a physical character-
istic of some biochemical* that for terrestrial use sites would
alaost preclude the potential for aquatic exposure and would reduce
the likelihood for residues on food or feed crops and residues on
terrestrial animal food, e.g., vegetation, invertebrates.
2. Maximum hazard testing. The concept of maximum hazard testing
is used in both the toxicology and nontarget organism sections.
The Agency includes information in Tier I that reflect a maximum
hazard approach to testing. The concept of maximum hazard testing
is that the most challenging exposure in terms of the treatment
dose or concentration, route of administration, and the age of
test animals is used in the first tier of testing. Using this
approach/ the Agency believes that negative test results from
testing under this approach would provide a high degree of con-
fidence that no adverse effects would be likely to occur from
the use of the biorational pest control agent.
3. Tier testing acheme. Four of the major section series, Residue
Chemistry, Toxicology, Nontarget Organism Hazard, and Environmental
Pate and Expression, use a tier testing scheme to ensure that only
the minimum data necessary to make a scientifically sound regulatory
decision are developed. This scheme eliminates the need for sub-
aittal of extensive data for those pesticides that are determined
to be safe on the basis of Tier I data. The Agency believes many
biorational pesticides will require only Tier I testing. The tier
testing scheme is discussed in detail under paragraph ZZZ D of this
Discussion.
B. Summary of Major Issues.
Major issues are identified and discussed in various sections
throughout this Discussion. The following list itemizes each issue
and indicates the location of each corresponding explanation in
this Discussion.
- State of the art in safety testing protocols for miczobial
agents: V.B.I.; VII.B-1.2(f); VIZ.B-2.3U)) YII.B-3.3{b);
- Potential hazards unique to microbial pest control agents:
V.B.4(a)6(b); VZI.B-1.2(e); VI.B-3.3(d)-(f);
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- Residue analysis procedure* and tolerance setting for microbial
•gents: VI.B.;
• Unresolved questions pertaining to Tier Z test protocol for
nontarget organisms: VII.A-2.3(a)4(b); VII.B-1.2(a)-(d); VII.B-
2.3(b)-(h); VII.B-3.3(a);
• Dse of in vitro testing for nontarget organism hazard assessment:
VII.B-2.3(a); VXI.B-3.3(c);
- Promoting biorational pesticides by modifying label claim
requirements: XX.
IV. PRODDCT ANALYSIS
A. Biochemical Pest Control Agents.
The product analysis data for biochemical pest control agents
151-10 through -18 of this subdivision) closely parallel those
•or conventional chemical pesticides as specified in Subdivision D
(§§ 163.60-1 through 64-1.) Both Subdivisions 0 and X of these
guidelines solicit detailed information on the procedures by
which the active ingredient is produced, and the techniques used
to ensure a uniform or standardized product. Refer to §§ 151-
11(a)(ii), (iii), (iv) and 151-12. If the standardization tech-
niques include methods of bioassay, then these methods should
be described. The Agency is particularly interested in the ingre-
dients which may be toxic or sensitizing to humans or other nontar-
get species.
B. Microbial Pest Control Agents.
The product analysis data for microbial pest control agents
under §§ 151-20 through -26 of this subdivision, to some extent,
parallel those for conventional chemical pesticides as specified
in Subdivision 0. However, due to the unique nature, composition,
and node of action of the microbial agents, there are some impor-
tant 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 $$ 151-20
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through -26 generally reference the corresponding section in Sub-
division 0 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 biorational pesticide used in the field is the same as
that which was tested for safety.
V. TOXICOLOGY
Biorational pesticides affect pest populations by controlling
physiological processes, by altering behavior, by competing for
space and nutrients, by parasitizing and lysing the pest, or by
replicating in an infective process to cause disease so that the
pest is destroyed. Compared to most conventional pesticides, they
act in very small amounts when used in the field. However, in
contrast to the usual concentrations of these agents found in the
environment, BOBS of these agents will be distributed or strategi-
cally placed in the environment in relatively high amounts for short
periods of time. The testing for registration of the product and
the kinds of data, developed must be sufficient to allow scientific
experts to assess the potential hazards associated with the use of
biorational pesticides.
The Agency was greatly assisted in developing the guidelines
for human hazard evaluation testing for biorational pesticides by
work performed by the American Institute of Biological Sciences
(AIBS) under Grant No. R806461. The report from this grant (Human
Hazard Evaluation Testing for Biorational Pesticides) served as the
basic document for this toxicology section.
The major concerns of the Agency regarding biorational pesti-
cides with respect to toxicology are:
- (1) "infactivity" - the potential for the microorganism to sur-
vive and replicate in a human host. Related concerns include
persistence, invasiveness, colonization, and other host-parasite
interactions.
- (2) "virulence-toxicity" - the potential for direct injury at
the cellular, tissue, or organ level. Included are -the long
term effects associated with oncogenicity, careinogenicity, and
teratogenicity.
- (3) "hypersensitivity" - an immune response leading to an
abnormal sensitivity. Serious reactions include allergies and
anaphylaxis.
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These concern* must be addressed in terms of the potential
impact of these agents on the population as a whole, not only with
respect to the normal individual but also with respect to persons
with altered defenses who might encounter these agents, and who
represent a subpopulation at higher risk. At present, the viruses
are of particular concern because they generally exhibit a greater
incidence of genetic change than other living forms. More inten-
sive testing related to this characteristic, therefore, is indicated
for the viruses.
The test batteries should provide negative data or data meet-
ing specified limit criteria to assure the Agency that adverse
effects would not result frcn the use of the agents. Because this
conclusion must ordinarily be reached in order to register a bio-
rational agent and because of the economics involved, the most
desirable and practical approach to testing involves the tier
concept. In general, testing beyond the first tier would be
instituted when data demonstrating the potential for adverse
effects are generated in the first Tier. It is recognized that
for some biorational pesticides there are no well-recognized and
standard test methods for assessing the toxicological.hazards to
mammals. Moreover, the testing method employed may vary among
pesticide products simply because of the characteristics of the
active agent. For example, it may be virtually impossible to
conduct experiments exposing animals to aerosols of some fungal
preparations because of the size of the fungal spore or mycelial
element, the recovery rate for the test organism, the nature of
the formulated product, or the concentration of the active material
in the formulated product.
In the development of biorational pesticides, many problems
related to hazard evaluation undoubtably will be encountered simply
because the field is new. Much innovative research is required,
and the standard methodology used with conventional pesticides is
not easily adapted to some of the biorational agents. When problems
arise, the registrant is urged to discuss the matter with the Agency
so that alternative methods and protocols can be considered prior
to the actual conduct of the tests.
Tables 3 and 4 under ( 152-1 summarize the testing for
biorational agents using the tier approach. Details are provided
in §§ 152-1 through -53 of the guidelines.
Biochemical Pest Control Agents.
1. Introduction. Two basic assumptions served as the foundation
upon which the biochemical testing regimen for evaluating human
hazard potential was developed. First, an exemption from testing
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based on extremely low exposure alone is not permitted by the
Agency; second, standard chemical toxicity testing, as for the
registration of conventional organic chemical pesticides, will
generally apply to biochemicals considered for use as biorational
pesticides.
2. Approach. The following general characteristics of biochemical
pest control agents and their uses were considered in developing
the safety evaluation testing protocols:
- (1) These compounds are generally not innately toxic, and their
pesticidal action is not the result of target organism toxifica-
tion.
- (2) Zn general, semioehemicals will be used in the field at
very low use rates compared to conventional chemicals. Hormones
or enzymes may be used at higher rates than semioehemicals, but
use rates, in terms of active pesticide ingredient, will also
generally be low. Therefore, human exposure to these products
will be generally lower than human exposure to conventional
pesticides.
- (3) Zn many instances (for semioehemicals especially), the site
of physiological activity in the target pest will have no analo-
gous site in non-target organisms or mammalian systems. Even in
the case of enzymes, substrate sites may be species specific,
and transport of enzyme in an active form to an intracellular or
internal nontarget organ site following dermal* oral, or inhala-
tion exposure is of low probability.
- (4) The biochemicals to be considered will be of known structure
and, in most instances, information on the metabolic pathways
for their synthesis and degradation will be available. Predict-
ability of lack of formation of potentially toxic metabolites is
highly probable: for example, degradation products of enzymes
are generally without significant toxic potential. Although
some peptides may be physiologically active, any toxicity
associated with these breakdown products would be reflected in
actual tests performed in vivo.
Pheromones isolated and purified from a siagla Insect species
may consist of a number of organic compounds, usually structurally
related with similar properties. Zf the degradation products of
the pheromone mixture have been chemically and physically charac-
terized, and display little or no evident toxicity on the basis of
available data, then it would not be necessary to conduct toxicity
testing for each individual component of the naturally occurring
mixture.
Conceivably, it may be desirable to combine several pheromone
mixtures into a single product in order to broaden the target
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species, to enhance stability, or to provide other benefits to the
user. In such instances, each pheroaone complex should be toxic-
ologically tested for possible adverse effects. In addition, the
formulated product containing mixtures of complex pheroaones may
be tested.
As with all biorational pesticides, the concept of maximum
hazard testing is used early in the tier testing regimen. The
concept of maximum hazard testing is that the most challenging
exposure in tens of route of exposure, species and age of test
animal, dose administered, and similar factors, will be used in
tier I to identify any potentially hazardous agents. The Agency
recognizes that the use of the •**'''"V" hazard testing approach
will require flexibility in determining the appropriate and feas-
ible do*e(s) and routed) of administration.
If the studies conducted in Tier I provide only negative data,
no further testing is indicated. If, however, a potentially adverse
effect is detected in maximum challenge experiments, the tests
proceed through the tiers until the actual hazard of the agent can
be evaluated and quantified.
In the first series of tests, data regarding acute toxicity,
irritation, hypersensitivity, mutagenicity, and effects on cellular
immune response are generated. Subsequent tests include subchronic
and chronic exposures of animals, teratogenieity, oncogenicity and
additional tests on autagenicity and cellular immune response.
Appropriate controls must be tested to ensure that any toxicity
or lethality is due to the presence of the active ingredient in the
formulated product. Insofar as possible, the control for the test
material should be identical to the formulated product except that
it should be biologically inactive. Treatment by mild physical or
chemical agents to inactivate the product or substitution of a
similar but innocuous and inactive chemical for the active compound
may provide a suitable control preparation for test.
3. Testing to address the major human safety concerns. The major
concerns of the Agency for humans and domestic animals regarding
the use of biochemicals as biorational pesticides are their acute
toxicity, possible irritation or sensitization, and potential for
mutagenic, teratogenic, or oncogenic activity, and effects on the
cellular immune response system.
Acute toxicity determinations should be performed using the
manufacturing use product, the technical grade of the active in-
gredient, and/or the formulated end-use product with test animals
exposed by the oral, dermal, and inhalation routes. These acute
toxicity tests-, when supplemented with tests-on irritancy; -hyper-
sensitivity, and cellular immune responses, should provide the
necessary information to assess the toxicity hazard.
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The potential for dermal and ocular irritancy, particularly of
the formulated product, needs to be assessed in order to protect
people handling the product. The following studies are presently
considered sufficient to characterize this potential:
- (1) Primary skin irritation tests in laboratory animals by use
of patch tests on intact and abraded skin, and, in certain
instances, tests of irritation of the eye; and
- (2) Observations in exposed people during laboratory testing,
pilot production, handling, transport, and field trials of the
product.
A biochemical pest control agent, especially a protein or
hapten, may induce allergic responses. In order to develop ade-
quate label precautions for handling the material, the registra-
tion applicant should investigate the biochemical's allergenicity
potential. The studies include sensitivity testing in experimental
animals, and the species selected should be known to give some
prediction of the potential of the product or its ingredients to
cause immune disorders. In addition, the registrant should report.
and submit any observations of such effects as skin sensitivity,
respiratory distress, or allergic symptoms in people handling the
material during its development or production.
Many well-established in vitro immunological techniques are
available that can provide information as to whether a substance
affects the immune response. .Such in vitro tests often correlate
well with _in vivo results. Positive correlations between _in vitro
and in vivo results have been observed with a wide variety of anti-
metabolites (i.e., protein and nucleic acid-inhibiting substances),
x-irradiation, and corticesteroids. For example, almost all agents
which suppress the antibody-forming ability of lymphoid cells in
vitro have siaillar effects in vivo.
The Agency is providing a tier system to ascertain the poten-
tial immunologieal hazard of the biochemical agents for humans.
This testing system measures the effect of the agent on immuno-
competence with both in vivo and ^n vitro tests including measure-
ments of blood cell count, leukocyte responses (T and B cell num-
bers), functional activity of blood leukocytes, swcrophage number
and function, serum protein levels, antibodyforming activity, and
cellular immune responses.
Information generated by the immune response tests will also
be of value as a screening procedure for carcinogenicity, since
many isnunomodulating agents are also carcinogens. The testing
scheme described above reveals not only carcinogens which affect
lymphoid cells -per-se, but also -agents which fceve -other carc-inogetU*
activity.
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4. Evaluating potential for oneogenic and mutaqenic effects.
These guidelines provide a comprehensive testing scheme to assess
the potential oncogenic and mutagenic effects of each biochemical
pest control agent. This scheme consists of three parts:
- (1) Microbial assays in Tier I and short-term mammalian assays
in Tier II;
- (2) Cellular ianune response studies in Tiers I and II; and
- (3) An oncogenicity study in Tier III.
All three parts of the testing scheme could apply, depending on
the biochemical agent, its use pattern, expected human exposure,
and results of certain other studies.
The approach recommended by a panel of American Institute of
Biological Sciences (AIBS) scientists would be to require only a
microbial bioassay (Ames test) and cellular immune response studies
in Tier I, to require the oncogenicity testing in Tier II, and to
require a chronic (oral, dermal, or inhalation) study in Tier III.
The approach proposed in the September 29, 1980 draft of these
guidelines, and subsequently endorsed by the FIFRA Scientific
Advisory Panel (SAP) in October, 1980 was to require:
- (1) A battery of five mutagenicity studies in Tier I consisting
of:
- (a) A bacterial assay for reverse gene mutation (Ames
assay};
- (b) A mammalian cell point mutation assay in vitro;
- (c) A prophage induction assay in lysogenic bacteria;
- (d) A mammalian in vivo cytogenetic assay, and;
- (e) Either a DMA damage/repair assay, or an assay for
mitotic recombination.
- (2) Cellular immune response studies in Tiers I and II; and
- (3) Oncogenicity testing at Tier II or III.
After careful consideration of the SAP's comments and the
written and oral comments from industry, the Agency decided to
include a somewhat modified approach to mutagenicity testing as
described above. This modified approach is consistent with the
SAP's comments and also responds to industry's request to reduce
the economic impact of the mutagenicity testing requirements.
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The cellular immune response studies will serve as screens
that identify potential carcinogenic agents, as well as provide
information on effects of the immune response system. The second
set of cellular ianune response studies would be located in Tier
II.
Testing for oncogenicity in mammalian species would apply
only if the following conditions are met:
- (1) If the biochemical agent produced, in Tier II subchronic
studies under $$ 152-19 through -21, a morphological effect/
e.g., hyperplasia or metaplasia, in any organ that potentially
could lead to neoplastic change; or
- (2) If adverse effects suggesting oncogenic potential are
observed in the Tier II cellular iasaune response studies or
short term mammalian mutagenicity assays.
When appropriate, an oncogenic evaluation could be combined with a
chronic feeding study.
B. Mierobial Pest Control Agents.
1. Introduction. The Agency provides guidelines for the registra-
tion of four groups of microorganisms that may serve as pest control
agents: bacteria, fungi, viruses, and protozoa. The Agency will
develop guidance for testing other kinds of microbial agents (e.g.,
algae) as the need arises.
Hazard evaluations of microorganisms used as biological control
agents are not on as firm a basis as is the case with conventional
chemical control agents, simply because the field is relatively new
and the interpretation of laboratory data in the light of mammalian
hazard is difficult. Nevertheless, the Agency must use current
methodology to assess the potential hazards associated with the use
of microbial pest control agents. Much information is generated
during the course of research and development of a microorganism as
a microbial pest control agent, and industry is expected and, in
many areas, required, to submit this information MM -part of the
application for registration. The Agency looks to researchers in
indus'-ry, academia, and Federal and State agricultural organizations
for comment and information on the design and development of more
relevant testing methodology so that specific issues of concern
regarding the registration and use of the microbial agents can be
addressed.
Accurate identification of the organism, as called for in
§ 151-20 of these guidelines, and its relationship to known human
pathogens is essential before meaningful mammalian safety tests can
be conducted and evaluated.
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Moreover, information on stability, persistence, susceptibility
to antibiotics, optimum conditions for growth in vivo and jj vitro,
recovery from host tissues and the natural environment, reliability
of assay and test methods, resistance to chemical and physical fac-
tors, metabolic activities, knowledge of structural, biochemical,
and other determinants involved in the infectivity-pathogenicity
process, and certain other characteristics of the microorganisms
are important as a basis for hazard evaluations* Information on
the mode of action of a microorganism as an insect or plant
pathogen can be analyzed and evaluated in terms of a possible
identical or similar mode of action in mammals.
2. Approach. The safety evaluation most be developed for each
group of microorganisms used as pesticides by taking into account
the characteristics of the organism and its proposed use.
The tier approach is used to develop safety testing in a
stepwise manner employing »y»<«m«it challenges and proceeding, if
necessary, with lesser challenges and other tests until the actual
hazard of the agent can be quantified accurately.
The concept of maximum challenge experiments is useful when
approaching safety testing of mlcrobial agents because a candidate
agent submitted for evaluation and testing is likely to be either
totally innocuous to nontarget organisms or to be an opportunistic
pathogen. It is clear that certain microbes known to be mammalian
or human pathogens most probably will not be seriously considered
for commercial development and will never reach the point of devel-
opment requiring extensive safety tests. If the organism proves
totally innocuous by the maximum challenge tests, further tests
would be unnecessary. If a potentially adverse effect is detected
in maximum challenge experiments, the tests proceed through the
tiers until the actual hazard of the agent can be clearly evaluated.
Several important general properties of microbial agents have
relevance for predicting human hazard. These include the ability to
mutate, to form different virulence factors such as toxins and en-
zymes, and to otherwise change their spectrum of pathogenic!ty. In-
formation on the ability of the organism to survive and replicate at
mammalian body temperature is Important to allow an evaluation of
potential mammalian pathogenicity. Host range or species specificity
information, if it exists, is important in assessing the hazard of
the agent for mammals. It is also particularly useful in the eval-
uation of safety tests if methods for recovery of the organism in
the presence of other similar organisms have been developed or are
available.
In the development of these guidelines, it was recognized that
tie specitic protocols could be provided at this time for the isola-
tion and enumeration of the various microorganisms. These protocols
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will differ depending on the genus and species under test. In
time, it is Anticipated that specific protocols can be developed
and then adopted by mutual consent of expert microbiologists for
various genera of microorganisms that have proven useful as pest
control agents.
3. Considerations for animal studies. The following discussion
illustrates a number of important aspects of animal testing with
microbial agents that should be considered by prospective regis-
trants. Comments on these aspects are invited so that the Agency
can determine their importance and relevance to the, tests. In the
conduct of animal tests, as much information as is feasible should
be obtained in each experiment so that the data provided will
permit well-considered judgment* to be made regarding the hazards
of use of the microorganism. For example, in LD50 tests informa-
tion on gross pathology, dissemination, replication, and survival
of the microorganism in animal tissues, organs, and fluids should
also be obtained. Clinical signs of illness such as elevated
temperature, unkempt appearance, altered feeding habits, weight
loss, various forms of distress, depression, and other similar
effects are also important observations, even though the animal
may recover completely from the exposure. Animal excreta should
be examined for the presence of the microorganism. An adequate
number of animals should be exposed to the microbial control agent
so that periodic sacrifices can be made to examine tissues and
organs for gross pathology and the presence of the control agent.
The organs or tissues receiving the initial challenge dose should
receive close examinations for pathological changes, i.e., the
lungs and upper respiratory tract for animals exposed to the
aerosol, the intestinal tract for animals receiving the oral doses,
and the skin of animals tasted by the dermal route. Qualitative
and quantitative measurements should be made to obtain evidence
for survival and multiplication of the microorganism.
To study the infactivity of microbial agents, the urine and
feces of test animals may be collected and examined for the presence
of the inoculated microorganism. If present, serial passage of
recovered isolates should be carried out with a standard serial pas-
sage procedure. The infactivity characteristics of the serially-
passed isolates should be compared to the infectivity characteristics
of the original test strain. Enhancement of virulence, survival,
invasiveness, toxicity, or resistance to clearance mechanisms are
indications of increased infactivity.
Overt symptoms of infection and disease related to pathogenic
effects of challenging organism* will be considered as presumptively
grave effects if observed in Tier I testing. However, the Agency
recognizes that simple organism survival of the challenging organism
is not generally regarded as a potential adverse effect, and that
some survival over short time periods is an expected biological
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phenomenon. Studies have demonstrated that the mammalian system
challenged with a non-proliferating organism will require some
reasonable time period to cleanse itself of the challenge dose.
(This time period is termed 'period of clearance".) The period of
clearance will vary, depending on the nature of the organism, the
numbers in the challenge dose, the site of administration, and
the immunocompetence of the animal. Survival of the test organism
at the site of administration or at distant sites following adminis-
tration for a period of only a few days will be considered as a
negative finding.
The Agency considers that attempts should be made to establish
mammalian LO50 values for all potential routes of human exposure,
namely oral, dermal, and inhalation. Preliminary probing types of
experiments would be conducted initially; that is, a few animals,
with suitable controls, would be exposed to maximum feasible con-
centrations of the product. If no animals succumb, obviously no
LD50 estimate is possible and no further testing is required. If
sufficient animals show a lethal response, then appropriate step-
wise dilutions should be tested to ascertain the LD50 value.
Dissemination rate, recovery rate, cloud concentration, and
respiratory rate are the principal factors needed to calculate the
dose administered by the respiratory route. Data from simple
dynamic aerosol chambers that permit studies on whole-body or nasal-
area-exposed animals may not yield this information unless the dis-
secinatory device is capable of creating aerosols in the 5 micron
range (pesticide particle size). A small aerosol size is necessary
to assure that the microorganism reaches the alveolar spaces thereby
providing the may!""!"1 potential for illness or death. Larger par-
ticles (microorganisms) may not reach the deeper recesses of the
lungs as they usually lodge in the upper respiratory tract and are
oore readily cleared. It is also important that recovery data on
microorganism viability both before and after dissemination from
the device be provided so that doses can be estimated.
Appropriate controls must be tested to ensure that any illness
or lethality is due to the presence of the active ingredient in the
formulated product. Suitable controls for products containing
replicable organisms would be "killed" preparations. Insofar as
possible, the control for the test material should be identical to
the formulated product except that it is biologically inactive.
The manufacturer is generally best able to provide such material
for the study.
In animals challenged by any of the various routes, an assay
for antibody production should be performed by the most appropriate
method that takes into consideration the animal species and the
microbial agent involved in the test. This requirement necessitates
pre-bleeding bF"tfie~ animals for base-line titers and additional
bleedings for serum collection at the appropriate time following
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animal exposure. Such antibody tests may screen animals for previous
exposure to the challenge organism and also demonstrate the ability
of the animal to produce antibodies in response to the challenge
dose. This test may also reflect the survival and replication of
the microorganism in the host.
Since microorganisms and their spores are composed of protein,
protein complexed with other biochemicals, and other antigenically
active materials, it is very likely that hypersensitivity reactions
of the non-immediate type will be achieved in experimental animals.
This indicates the need for data on the development of hypersen-
sitivity of the non-immediate type in animals; in these studies
the technical control agent preparation Is tested.
The Agency would also examine information on any hypersen-
sitivity or allergic reactions that were experienced by personnel
involved in the research or development of the biological control
agent. The ease with which hypersensitivity reactions are achieved
and the severity of these reactions in experimental animals should
provide a basis, in conjunction with the reports from industry
regarding hypersensitivity experience in workers, for evaluating
the hazard of hypersensitivity fox humans and, possibly, other
mammals, associated with the use of the biological pest control
agents.
4. Major concerns, (a) General. As previously stated, the major
concerns of the Agency regarding hazards of use of replicating
biological pact control agents affecting humans and other mammalian
species are infectivity, allergenlcity, irritancy, and toxlcity.
All candidate agents for registration must be tested for potential
hazards. The amount of testing varies, however, for different types
of agents based, in part/ on the historical record of related
organisms that showed a potential for hazard.
(1) Infectivity. The evaluation of potential microbial
infectivity in mammals is based on information on the nature of the
microbial agent and on data derived from studies especially designed
to assess this specific hazard. In spite of the special design of
infectivity tests in experimental animals, there is still some
uncertainty in extrapolating results from experimental animals to
man. The use of maximum challenge tests in a selected variety of
test animal species is used to provide an adequate -degree of con-
fidence in the data. Much of the data characterizing the micro-
organism with respect to species specificity,'Infection processes,
hosts and substrates, susceptibility to chemical and physical
agents, and other similar factors* aids In evaluating the hazards
of using the agent for part control.
Section series 152 covers the submission of information on
survival, distribution, replication, and gross pathology in test
mammals following administration of the agent at high dose levels
by various routes.
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(2) Allergic responses. There is a definite possibility that
a aucrobial pest control agent might induce allergic responses in
nontarget animals and humans; this conclusion is based on the well-
documented allergic responses elicited by the cell components of
fungi, bacteria, and protozoa. Although it is possible that a
virus could induce such a response, it is more likely that other
constituents of the viral technical control agent formulation
would serve as allergens. Zn order to recommend adequate pre-
cautions in handling the material, its allergenicity potential
must be investigated in test animals. In addition, information is
also needed regarding any observations of skin sensitivity,
respiratory or other allergic symptoms in people handling the
material during development, production, and application.
(3) Irritaney. The potential for dermal and ocular irritaney,
both of the technical and of the end-use formulated product, needs
to be assessed as part of the primary battery of safety testing in
order to adequately protect people handling and using the product.
The following studies are presently considered sufficient to
characterize this potential:
- (1) Primary skin irritation tests in laboratory animals by use
of a patch test to intact and abraded skin, and, in certain
instances, tests of irritation of the eye; and
- (2) Observations in exposed people during laboratory testing,
pilot production, handling, transport, and field trials of the
product.
(4) Toxicity. The toxicity potential may be associated
with microbes which produce toxins related to their mode of action
in target organisms, or metabolic products unrelated to their mode
of action as biological pest control agents. Moreover, since prod-
ucts used as biological pesticides will be produced using fermen-
tation technology or whole animal technology, very pure preparations
of active ingredient (agent) in the form of the technical product
are not expected. To test for the possibility of toxic components
other than the replicating agent in relatively impure preparations,
as well as to characterize the human toxicity potential of prepara-
tions which contain known toxic components for target organisms,
it is necessary to perform, minimally, several acute toxicity
studies on both the technical and end-use formulated products.
These acute studies include acute oral and dermal tests and skin
and eye primary irritation tests that are located in the Subdivi-
sion P guidelines.
(5) Immune responses. The Agency has provided guidelines for
testing and data submission concerning toe-possible influence of
biological pesticides on immune responsiveness and other immune
parameters. Many assays are available to assess the effects of
these agents on Immunity.
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Well-established immunological techniques are available that
can provide information as to whether a substance affects the immune
response. A wide variety of antimetabolites such as protein- and
nucleic acid-inhibiting substances, x-irradiation, and corticosteroids,
markedly suppress antibody formation in vitro. Such in vitro tests
often correlate well with in vivo results. For example, almost all
agents which suppress the antibodyforming ability of lymphoid cells
in vitro have aim Liar effects in vivo. It appears likely that
these tests may provide some information on the carcinogenic activity
of these agents, particularly with regard to effects on lymphoid
cells.
(b) Specific concerns. (1) Bacteria. If, in the infactivity
studies, a bacterial agent shows evidence of survival and replication
in the test animal, the possible increased infactivity of the micro-
organism would obviously be of concern. Serial passage studies
would be the next logical testing step so that new isolates could
be compared to the original strain for enhancement of virulence,
survival, invasiveness, toxicicy, or resistance to clearance mech-
anisms. If such studies indicate poor genetic stability, further
animal studies would be indicated.
(2) fungi. AS with bacteria, virulence enhancement is also a
concern with fungi. Although relatively few fungi cause infections
in humans with any regularity, the ability of certain fungi to
adapt to the environment and grow under unfavorable conditions
creates concern for the safety of humans and other mammals that may
come in contact with fungi used as microbial pest control agents.
Some fungi have, a very wide range for growth, although the optimum
temperature for a typical non-pathogen is generally much lower than
the body temperature of mammal*.
The degree of specificity shown by entomogenous fungi is quite
variable. Some can grow only in insects, whereas others also can
utilize substrates found in the soil and artificial media. Some
fungi that grow on plants need nutrients supplied by the plant in
order for the fungal spores to germinate.
Great diversity is often shown by fungi with respect to hosts,
growth temperature, spore germination requirements, enzyme composi-
tion, resistance to physical and cheaical conditions, nutrient
requirements, dose necessary for infection and several other fac-
tors. Because of this diversity, a full characterization of the
fungus, as outlined by $$ 151-20, 22, and -26, aids in assessing
the hazards of use of the fungus as a microbial pwrt control agent.
The hazards of use undoubtedly will vary from one fungal species
to another.
(3) Viruses. The potential lack of genetic stability of
viruses with respect to species specificity is of concern to the
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23
Agency. To test the capacity of « virus to infect and interact with
nontarget species, a series of tests are proposed involving tissue
cultures of cells of both hunans and nonhuaan origin. The tests
invlude -observations on gross morphological changes (cytopathic
effects), inhibition of cell division, bioassays of culture fluid,
decay of input virus, and potential appearance of viral proteins
and nucleic acid.
While there is no evidence to indicate that any currently
registered viral pest control agents represent a hazard to humans,
a critical review of the literature indicates deficiencies in test
systems utilized, in the )cinds of data collected, and in the
verification of testing results, such that the issue of potential
hazards has not been completely resolved.
In recent years, new technologies have been developed that
allow for a more precise evaluation of the potential for genetic
instability in viruses, for example, the potential for genetic
change can be assessed in the laboratory by several techniques.
Tissue culture cells of human and nonhuaan origin provide a sensi-
tive means for testing the capacity of a virus to infect and in-
teract with non-target species. Results from these tests, along
with acute high dose infectivity and replication studies and stan-
dardized tests on irritation, hypersensitivity, and cellular immune
response, will provide an appropriate data base for assessing the
potential capability of viruses used as pesticides to cause adverse
effects in humans.
It is important to note that special precautions are necessary
to ensure that formulated products contain the desired infectious
viral agent and are not contaminated with additional entities that
could pose a hazard to hunans. It is likewise important that the
virus that is tested, whether in the formulated product, in the
technical control preparation, or in its purest infective form, be
identical to the virus in the commercial product. Because viruses
are infectious agents that are widely distributed in nature and
will be derived from crude material such as insects or tissue cul-
ture, it is possible for the viral control agent to be contaminated
with other agents. The virus should be fully characterised as
prescribed in the section dealing with the product analysis of
microbial pest control agents (§§ 151-20 through -27).
In the course of development of an entomopathogen as a micro-
bial pest control agent, the research and development activities
could provide knowledge of structural, biochemical, and other
determinants Involved in the infectivity-pathogenicity process.
Information on the mode of action of the microorganism as an insect
pathogen can be analyzed and evaluated in terms of its possibly
identical or -similar mode of action -in mammals.
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24
(4) Protozoa. These microorganism* present several concerns
;or mammalian safety because of their ability to survive and to in-
vade a wide spectrua of hosts, frequently including alternate hosts.
Many protozoa are able to survive and multiply in hosts at mam-
malian body temperature. Further, scsw protozoa are able to pene-
trate the gut or the intestinal tract and various body tissues
mammals• Most damage to the insect host is caused during the vegeta-
tive, shizogonial period of development when the protozoa destroy
host tissues.
Protozoan infection in insects results from the ingestion of
protozoa contaminating the insect's food* During the use of protozoa
as microbial pest control agents, infection in hnsisns could occur
following ingestion, but also could occur by respiratory or dermal
routes of exposure. For this reason, all of these routes are to be
investigated in experimental animals. For instance, if there is any
evidence of oral infactivity in the acute test, a more extensive
feeding study should more clearly define the hazard.
Since protozoan products used as biological pesticides vill be
produced from infected insects or possibly from tissue cultures,
relatively pure preparations of active ingredient (agent) in the
form of the technical product are not expected. To check for the
possibility of acutely toxic components in relatively impure pre-
parations other than the replicating agent, it is necessary to per-
form, minimally, several acute toxicity studies on both the techni-
cal and formulated products, including acute oral and dermal tests,
and skin and eye primary irritation tests as required in Subdivi-
sion F.
5. Literature review and discussion of potential hazards, A few
species of each of the four types of microorganisms (bacteria,
fungi, viruses, and protozoa) have been investigated extensively
for use as microbial pest control agents, and XPA registrations
or experimental use permits exist for each of the four types of
agents. A brief discussion follows on the various types with
emphasis on the major mammalian hazard concerns.
(a) Bacteria, laboratory and cosmtercial preparations of
Bacillus thuringiensis have been subjected to numerous evaluations
for possible toxicity in vertebrates since 1957. Summaries of the
studies conducted to determine the hazard to humans of use of bac-
terial agents as insecticides have been published.
(b) Fungi. EPA sponsored an American Institute of Biological
Sciences workshop in 1975 to elaborate principles and criteria for
establishing the safety of fungal agents (AIBS, 1975). There are
nearly 100,000 species of fungi, but perhaps less than 50 cause
human infection with any regularity, and clearly not any of these
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25
would be considered for use as a pesticide. One cannot ignore, how-
ever , the ever-increasing li»t of obscure fungi of pathogenic poten-
tial, even though the numbers of cases attributed to any one species
remains low. For example, within a period of only two decades,
two insect-associated fungi have been recognized as pathogenic for
man, viz. gntoaophthora coronata, causing rhinophycomycosis, and
aasidiobolus meristosporus, the etiological agent of subcutaneous
phycornycosi» (Greer, 1977). Both organisms are thought to enter
the host by trauma and remain localized at the site of injury;
i.e., neither has displayed any tendency to become disseminated
systemically.
Even more clearly associated with trauma are the etiological
agents of mycetoma, a condition that tends to remain localized, and
appears unrelated to predisposing factors such as isBunosuppressive
therapy. The overwhelming majority of cases are caused by a rela-
tively few species (fimmon* et al., 1977), but it should be con-
sidered that any fungus having the capacity to grow at the temper-
ature of the human subcutaneous tissue may have this pathogenic
potential. Similarly, there should be consideration of the poten-
tial for an agent to cause subcutaneous cysts (fissons et al.,
1977) The agents of both mycetoma and cysts are naturally resident
in the environment.
Another disease caused by a variety of soil saprophytes includ-
ing fungi and preceded by trauma is corneal ulcer formation. This
condition, however, appears to be a consequence of immunosuppressive
therapy, for fungal ulcers of the eye were virtually unknown until
ophthalmologists began treating wounds of the eye by topical appli-
cation of corticosteroids (Rippon, 1974). Many of the organisms
causing corneal ulcers cannot grow at 37«C or more, a characteristic
that allows screening for such agents.
Another disease that exemplifies the enlarging spectrum of
fungi capable of causing systemic disease in man is that of the
brain abscess caused by Cladosporium bantianum (trichoides). This
condition was unknown prior to 1952. The organism is found in
man's environment, has a predilection for the central nervous
system, and has an unknown mode of infection. The condition does
not appear to be linked to immunosuppressive therapy*
Testing for the pathogenic potentials described above is not
feasible in every instance. There is no suitable experimental
model for reproducing diseases such as mycetoma, cyst, and subcu-
taneous phycornycc«is. There are, however, experimental systems
for recognition of agents capable of causing systemic disease,
especially those whose otherwise unexpressed pathogenicity could
be potentiated by immunosuppressivesj these are included in the
scheme of testing described below.
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26
Mycotoxicosis is a term used to broadly define toxic reaction
due to itvgestion of otherwise innocuous fungi or the metabolites of
such fungi. Ergot and mushroom poisoning are examples known since
antiquity. In the early 1960's, aflatoxins were discovered, first
from Asfrergillus flavus, but now from other species. Aflatoxins
are acutely toxic to many vertebrates. At least one is carcinogenic,
highly organ-specific, and causes liver tumors. There are now
known to be more than 100 myeotoxinsi thus, there is no single in
vitro test that can be used for detection of all of them. Chemical
analysis by chromatography has been widely used [thin-layer (e.g.,
Durackcva et al., 1976); high-pressure liquid (Engstrom et. al.,
1977)]. Enzyme-linked immunosorbent and iamunocytochemical tech-
niques have been tried (Lawellin et al., 1977), But biological
assays, (i.e., feeding), remain indispensable. (For a recent.
bibliography, see Maggon et al., 1977).
Perhaps a more important aspect of pathogenicity of the fungi
than either infectivity or toxicity is that of hypersensitivity
reactions. These occur mainly as respiratory problems, categorized
as either •immediate" or "nonimmediate" on an ianunologic basis.
Those of the immediate mechanisms are of the atopic variety exem-
plified by rhinitis and asthma; and those of the nonisnediate are
exemplified by allergic alveolitis (hypersensitivity pneumonitis).
These two major groups of hypersensitivity diseases are discussed
below. A third disease entity, which is closely related to the
second hypersensitivity category, is allergic bronchopulaonary
aspergillosis, a common cause of pulmonary eosinopbilia among
atopies in Great Britain. The most common offending organism is
Asperoillus fumigatus. Because of the known pathogenicity (includ-
ing infectivity) of many species of aspergilli, it is doubtful
that any would ever be under consideration for use as a pesticide.
Respiratory atopic allergies are a major health problem in this
nation and elsewhere. They are incited by many substances other
than fungi, but are commonly caused by fungi such as Cladosporiun,
He lainthospor iuffi, Altemaria, Curvularia» Pencillium, and Fusarium.
Since most fungi have the capacity to cause sensitization
reactions under ordinary circumstances, testing the capability of a
fungal pesticide to sensitize would not provide any additional in-
formation of value. Information, however, is needed concerning
the relative importance of an agent to elicit acute respiratory
symptoms among the population at risk.
Allergic alveolitis, essentially a hypersensitivity pneumonitis,
is presently the subject of intensive investigation* in a number of
institutions. This disease (which often provides descriptive clini-
cal names such as bird fancier's lung, farmer's lung, cotton worker's
lung) can be caused by a variety of materials including actlnooycetes
and fungi. One common feature of the disease, irrespective of the
agent, is a history of long and repeated exposure to aerosols of
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27
the offending agent. The precise mechanism is not clearly under-
stood, bur allergy of the non-immediate type is involved, and
patients usually have precipitating antibodies. The propensity of
my dust, including fungal spore dust, to cause hypersensitivity
can be determined in experimental animals by exposure to aerosols,
or by inoculation intranasally or intratracheally.
Exposure of test animals to aerosols of mycelial fragments or
spores of fungi has the advantage of approximating natural exposures.
In these test systems, however, it is difficult to administer a
predetermined dose deep into the alveoli and to confirm the actual
dose delivered. Xntranasal inoculation suffers from the same
disadvantages. Intratracheal administration by eannulation is an
alternative route of administration. Depending upon the animal
species, cannulation is mechanically simple, and delivery of dose
is reliable. Evidence of sensitization could be obtained by any of
several standard procedures. Refer to Bice et al. (1979) for an
evaluation of methods for pulmonary immunization.
(c) Viruses. More than 700 species of insects and several
species of mites are reported to have viral diseases. Several of
these viruses are registered a* pesticides with the Agency and, to
date, have not posed a threat to the health of humans or other
manuals. These insect viruses are considered to be host-specific,
most of them infecting only one host species or one insect group.
They usually reduce insect populations only when a certain density
of the insect population has been achieved, although the responsive-
ness to host density may be also dependent on the simultaneous
occurrence of other environmental and host conditions.
However, most viruses are able to produce latent infections
which, after stress, may suddenly manifest themselves as epizootics.
Although it is assumed that viruses are species-specific, it is
documented that viruses are capable of undergoing genetic change
through a variety of mechanisms, many of which are not well
understood. These mechanisms enable viruses to undergo genetic
interactions with each other and to incorporate genetic material
into the genomes of their hosts.
Because of the potential of viruses serving as biological
control agents, a great deal of research already has been conducted.
Most of the viruses affecting insects are either «ytcplasmic poly-
hedrosis viruses or baculoviruses such as nuclear polyhedrosis
viruses or granulesis viruses.
(d) Prototoa. Although many pathogenic proto*estt parasites
of insects exist, and some, such as gregarines and coccidia, may
hold promise for use as insect control agents, most efforts to date
have focused on microsporida. The genera Nosema and Vavria have
received special attention, although scattered information is
available for microsporida in other genera. Several points were
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28
considered in the development of the guidelines for evaluating
the human hazards posed by these organisms. First, these parasites
are widely distributed in nature, and large numbers are frequently
encountered in foodstuffs and water. Second, some, such as Mosema
algerae and Nosema locustae, have been the subject of numerous
laboratory investigations and large scale propagation without any
evidence of human disease. Third, serologic surveys of selected
human populations specifically designed to detect antibodies to a
variety of insect microsporidan parasites nave failed to reveal any
evidence of human infection (Chapupsky et al., 1972).
On the other hand, the protocols for evaluation of human
hazards.poeed by the use of insect microsporida must take into
account the following facts:
- (1) At least one microsporidian parasite affects a wide variety
of animals;
- (2) Classification of the organisms is at present an uncertain
and confusing issue; and
- (3) A few reports of human infections with microsporida do
exist.
These facts are discussed briefly in the next few paragraphs.
Eneephalitozoon euniculi is the cause of subclinical granule-
oatous meningoencephalitis and nephritis in rabbits, mice, rats,
and guinea pigs (Shaddock and Pakoo, 1971). It causes fatal en-
cephalitis in carnivores such as puppies (Shaddock et. al. 1978),
blue foxes (Mordstoga and Westbye, 1976), and.some species of wild
cats (Vavra et al., 1971), and the parasite has been reported
several times in nonhuman primates (Anver et al., 1972; Brown et.
al., 1973; and Siebold and Fussell, 1973). E. euniculi produces
more severe disease in iBBunologically compromised animals
(Bismanis, 1970). Classification of the genera of microsporida is
still an uncertain and confusing issue (Weiser, 1976). For about
10 years, £. cunieuli was classified within the genus Mosema
(Lainson et. al., 1964). Eneephalitozoon and Mosema are clearly
different in several important features (Pakes et. al., 1975), yet
the 10-year inclusion of these organisms in the same genus continues
to raise some concern about the potential role of Wesema as human
pathogens. The issue is not resolved, however, since one-vay
serological cross-reactivities between J£. eunieuli and two species
of Gluqea as well as Mosema algerae have been shown recently
(Nierderkorn, 1980). A few reports of human infection with micro-
sporida exist. Several reports were incorrect (Barker, 1974} or
have been retracted, but at least three cases have been documented
(Ashton and Wirasinha, 1973; Margileth, ct. al., 1973; and Matsu-
bayashi et. al., 1959}. The best documented case occurred in a
young ismunoineompetent child who succumbed with a variety of
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29
lesions including wide-spread mlcrosporidosis (Lainson et. •!.,
1964). The organism was named Nosema eonnori (Shaddock and Geroulo,
1979) bat its source was unknown. Indirect evidence that entomo-
pathogenic Hoaeaa nay infect nan includes the fact that Nosema
algerae (a mosquito pathogen) can replicate (though poorly) in
vitro in pig kidney cells at temperatures as high as 35*C, but not
at 37*C. A few replicative forms of £. alqerae have been found in
subcutaneous tissues following injection of spores into the tall
skin of nice. Mice injected subcutaneously in the tail produce
antibodies.
Literature Cited.
(1) Anver, M.R., N.W. Xing, and R.D. Hunt. 1972. Congenital
encephalltozoonosis in a squirrel monkey (Sainiri sciureus). Vet.
Pathol. 9:475-480.
(2) Ashton, N., and P.A. Wirasinha. 1973. Encephalitozoonosis
(noseaatosis) of the cornea. Br. J. Ophthal*"!. 57:669*674.
(3) Barker, R.J. 1974. The nature of Encephalitozoon brumpti
Coulon, 1924. J. Parasitol. 60(3):542-544.
(4) Bice, D.E., O.L. Harris, C.T. Schnizlein, and J.L. Manderly.
1979. Method* to evaluate the effects of toxic materials deposited
in the lungs on immunity in lung associated lymph nodes. Drug and
Chesu Toxicology 2:35-47.
(5) Bismanis, J.S. 1970. Detection of latent murine nose-
aatosis and growth of Sosema cuniculi in cell cultures. Can. J.
Mierobiol. 16:237-242.
(6) Brown, R.J., O.K. Hinkle, W.P. Trevethan, J.L. Rapper/
and A.E. McKee. 1973. Nosematosis in a squirrel monkey (Sa^-Jri
aciureus). J. Med. Primatol. 2:114-123.
(7) Chalupsky, J., J.P. Lenski, P. Bedraik, and J. Vavra.
1972. An attempt to demonstrate the connection between multiple
sclerosis and gneephalitozoon cuniculi infection. J. Protozool.
19 (Suppl.):76-77.
(8) Durackova, Z., V. Betina, and 9. Hemec. Vtf76. Systematic
analysts of atycotoxins by thin- layer chromatography . J. chromato-
graphy 116:141-154.
(9) tenons, C.W., C.H. Blnford, J.P. Utz, and K.J. Kwon-Chung.
1977. Medical Mycology. 3rd Edition. Lea and Febiger, Philadelphia.
tngstzon, G.W., J.L. Richard, and C.L. Cysewskl. 1977.
High pressure liquid chromatographic method for detection and resolu-
tion of rebratoxin, af la toxin, and other mycotoxins. Aor. and Food
Chea. 25:833-836.
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30
(11) Greer, D.L. 1977. Fungi of Phyomycosis. In E.H. Lennette,
E.H. Spaulding, and J.P. Truant (eds) Manual of Clinical Microbiology,
2nd Edition. American Society for Microbiology, Washington, D.C.
(12) Lawellin, D.A., D.W. Grant, and B.K. Joyce. 1977. Afla-
toxin localization by th« enzyme-linked immunocytochemical technique•
Applied Environ. Microbiol. 34:88-96.
(13) Lainson, R., P.C.C. Garnham, R. Killick-Kendrick, and
R.G. Bird. 1964. Noseoatosis, a microsporieidial infection of
rodents and other aniaals, including num. Br. Med. J. 2:470-472.
(14) Maggon, K.S., S.K. Gupta, and T.A. Vonkitasubraaanian. 1977.
Biosynthesis of *flatoxin. Bact. Reviews 41:822-855.
(15) Margileth, A.M., A.J. Strano, R. Chandra, R. Neafie, M.
Blum, and R.M. McCully. 1973. Disseminated noaematosis in an immune—
logically compromised infant. Arch. Ptthol. 95:145-150.
(16) Matsubayashi, M., T. Koike, I. Mikata, B. Takei, and S.
Bagiwara. 1959. A case of encephalitozoon-like body infection in man.
Arch. Pathol. 67:181-187.
(17) Niederkorn, J.Y., E. Weidner, and J.A. Shadduck. 1980.
Antigenie cross-reactivity among different microsporidan spores as
determined by immunofluorescence. J. Parasitol. 66(4):67S-677.
(18) Mordstoga, K., and K. Westbye. 1976. Polyarteritis nodosa
associated with nosematosis in blue foxes. Aeta Pathol. Microbiol.
Seand. (Sect. A). 84:291-296.
(19) Pakes, S.P., J.A. Shadduck, and A. Call. 1975. Fine
structure of Eneephalitozocn cuniculi from rabbits, mice, and ham-
sters. J. Protozool. 22:481-488.
(20) Rippon, J.W. 1974. Medical Mycology. The Pathogenic
Fungi and the Pathogenic Actinomycetes. W.B. Saunders, Philadelphia.
(21) Seibold, H.R., and E.N. Fussell. 1973. Intestinal
microsporidiosis in Callicebus moloeh. Lab. Anin. Sci. 23:115-118.
(22) Shadduck, J.A. 1980. Unpublished observations.
(23) Shaddu>.-k, J.A., R. Bendele, and G.T. Robinson. 1978*
Isolation of the causative organism of canine encsphalitozoonosis.
Vet. Pathol. 151449-460.
(24) Shadduck, J.A., and J.J. Geroulo. 1979. A simple method
for the detection of antibodies to Encephalitozoon cuniculi in.
rabbits. Lab. Anim. Sci. 29(3):330-334.
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(25) Shadduck, J.A., and S.P. Pafcas. 1971. Spontaneous
disease of laboratory animals which interfere with bioaedical
research. Encephalitozoonosis and toxoplasaosis. An. J. Pathol.
64:657-674.
(26) Vavra, J., x. Blarek, N. lavicka, X. Jtocxkova, S. Kalafa,
and M StehloJc. 1971. Nosematosis in carnivores. J. Parasitol.
57:923-924.
(27) Weiser, J. 1976. To the identity of the microsporidia
affecting man. Vestnik Ceskoslovenske Spoleenosti Zoologicke
40(2):157-159.
VI. RESIDUE ANALYSIS
A. Biochemical Pest Control Agents.
1. Background. A pesticide may not be used on a food or feed
crop, or may not be employed for a use which may reasonably be
expected to result (directly or indirectly) in residues in food or
feed unless a tolerance or an exemption froa 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 (FTDCA) (21 D.S.C. 346, 346* and 348). The residue chemistry
guidelines for tolerances/exemption are outlined in Subdivison 0 of
the guidelines. The procedural regulations for filing petitions for
a tolerance or an exemption from a tolerance are included in 40 CFR
180.7.
2. Approach. The full set of residue chemistry guidelines outlined
in Subdivision O may not always be applicable to biochemical pest
control agents for the following reasons:
- (1) Biochemical agents occur naturally in the environment or
are identical to naturally occurring biochemical* and have prop-
perties similar to their natural counterparts; and
- (2) Many biochemical agents are used at very low application
rates [i.e., 0*7 ounces (20 grams) active ingredient or less per
acre]; and
- (3) Past experience indicates that biocfae»icaV^g«nts are
relatively nontoxic. Consequently, the resulting residues of
biochemicals in food or feed would be very low and the potential
for adverse effects would be correspondingly low.
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32
Thus, the Agency believes that significant human dietary expo-
sure will generally not occur from the us* of biochemical*.
3. Ti«r Progression. In general, when no potentially adverse
effects are observed during the Tier X toxicity testing ($J 152-10
through -18), a biochemical would be exempted from the need for a
tolerance, providing it is applied at rates of 0.7 ounces (20 grans)
or less per acre per application. In this situation, the Agency
would waive the usual metabolism and residue data and would recom-
mend that an exemption from the requirement of a tolerance be made.
The full range of residue chemistry data as detailed in Sub-
division 0 would apply to:
- (1) All biochemical agents proceeding to toxicity testing beyond
Tier I (that is, to Tier ZZ or ZZZ, as described in §§ 152-19
through -29 of this subdivision); and
- (2) All biochemical agents to be applied on food or feed crops
at a rate greater than 0.7 ounces (20 grams) active ingredient
per acre per application.
These would include plant metabolism studies, residue data, and
analytical methodology. Za addition, depending on the level of
residue found in animal feed, the Agency may solicit data on animal
metabolism and feeding studies to determine the carryover of resi-
dues into meat, milk, poultry, and eggs. When appropriate, tole-
rances for the latter commodities would be necessary.
B. Microbial Pest Control Agents.
1. Approach. As with a biochemical agent, 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. Zn considering exemp-
tions from the requirement for tolerances, the Agency recognizes
that these agents do not necessarily pose the same potential hazards
as conventional chemical pesticides. Zn fact, certain character-
istics of many of these agents suggest that they eose relatively
less harard. These characteristics are listed below:
- (1) The efficacy of the agent often depends upon its ability
to replicate in the target pest vnlcfc Is not lifcelf to remain
on the crop after harvest.
- (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).
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33
(3) Certain environmental conditions such as sunlight, rainfall,
winds, humidity, and temperature often greatly reduce the
viability of the agent and, therefore, the residues of living
organisms axe apt to be small or relatively insignificant
shortly after application.
(4) When evaluated by the tier testing scheme, data supporting
currently registered microbial agents indicate that microbial
pest control agents would not likely pose a hazard to humans or
other
- (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).
2. Tier Progression. The Agency evaluates residue data for micro-
bial 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
§§ 152-30 through -39 of this subdivision. If Tier I toxicology
tests indicate no toxic or other harmful properties, then no resi-
due data would be indicated and thus a recommendation for an exemp-
tion from the requirements of a tolerance can be made.
3. Major 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. Zt may therefore be im-
possible to distinguish between natural and introduced microbial
populations and therefore be very difficult to establish and en-
force tolerances for naturally occurring microbial agents. The
Agency invites comment concerning the testing methods for estab-
lishment and enforcement of tolerances for naturally occurring
microbial agents.
VIZ. NONTARGET ORGANZSM HAZARD
The purpose of nontarget organism testing is to develop data
necessary to assess potential hazard of biorational pesticides to
terrestrial wildlife, aquatic animals, plants, and beneficial
insects.
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A. Biochemical Agents.
A-1. Terrestrial Wildlife and Aquatic Animals (General).
1. Approach. The Agency bases hazard evaluation of biochemical
agents OB tart* similiar to those required to support registration
of conventional chemical pesticides. However, the Agency is pro-
poning to reduce the number of Tier I tests and to modify test
design*. Tier I tests would be designed to determine whether LCSO
or LD50 values are above a specified maximum test concentration or
dose rather than to require that the I-CSQ or U>5o be determined.
This modified test design would apply if death was not observed at
the maximum concentration or dose.
The Agency believes that the reduced number of test* and th«
modified test designs are appropriate because:
- (1) Innate toxicity is not inherent to the nature and mode of
action of biochemical agents;
- (2) Experience indicates that most of these pesticides will be
applied at very low rates compared to conventional chemical
pesticides, thereby reducing likelihood of significant exposure
to nontarget organisms; and
• (3) ?ast experience indicates that meet biochemical pest control
agents are not acutely toxicj e.g., LC^g and LDgg values for
most biochemical agents are greater than 5000 ppm or 2000 mg/kg,
respectively (in avian species).
Both terrestrial wildlife and aquatic animal testing schemes
use some or all of the exposure criteria — low use rate, low
exposure formulation, nonaquatie use site, and high volatility —
to screen out those pesticides that qualify for reduced testing.
Volatility and its use as a criterion are explained as follows.
Volatility is a function of the vapor pressure of the biochemical
and its ability to adsorb or absorb to a substrate, e.g./ suspended
sediaent, feed, or soil. The Agency proposes that an estimate of
volatility be derived from the ratio of the substance's vapor
pressure and solubility in water. These data (vapor pressure and
solubility in water) are outlined in the product Analysis portion
(section series 151) of tliis subdivision. For the purpose* of these
guidelines, a biochemical pesticide is considered to have high
volatility i* the estimated volatility (H) is greater than 5x10~5
atm. m3/mole. The rationale for this criterion is presented in
part VIII of this Discussion.
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35
A more exact determination of volatility of the formulated
product is outlined as a Tier II teat (aee § 155-4), and may depend
upon the pesticide use site and adverse effects observed in Tier
I tests. Additional considerations concerning the exposure criteria
are explained later in this Discussion, as they apply to each
testing scheme.
In general, biochemical agents control behavior, growth, and/or
development of target organisms* Ideally, Tier I tests should be
capable of detecting adverse effects resulting from the primary
mode of action by which the pesticide would likely act on the non-
target organism. At the present time, however, the Agency does
not believe that it is appropriate to locate behavior and repro-
duction tests in Tier I for taro reasons. First, no obvious approach
or battery of behavior tests are universally suitable for pesticide
screening (Weiss and Laties, 1979). This is apparently a reflection
of the youth of this scientific discipline. Second, there are no
widely accepted short-term screening tests that indicate chronic
growth effects and/or developmental effects from pesticides on
terrestrial vertebrates.
The Agency provides the following criteria for determining
need for testing of biochemical pest control agents beyond the
first tier:
- (1) If signs of abnormal behavior are reported in Tier I tests
at levels equal to or less than the maximum expected environmental
concentration; or
- (2) If detrimental growth, developmental, or reproductive
effects can be expected, based on:
- (a) Tier I test data;
- (b) Available fate data from the product's research and
development;
- (c) Use pattern information;
- (d) Results of mammalian testing required in the Toxicology
section series 152; and
- (e) The phylogenetic similarity between target pest and
noatarget organism; or
- (3) If the maximum expected environmental concentration is
equal to or greater than 1/5 the LC50 values established in
Tier I terrestrial wildlife studies, or equal to or greater than
1/10 the LC50 or ECSO values established in Tier I aquatic animal
studies.
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36
In addition, both Tier Z and Tier ZZ tests would be indicated
1*:
- (1) The pesticide is to be applied directly to water; or
- (2) High use rates are proposed; and
- (3) The biochemical agents are not volatile.
A-2. Terrestrial Wildlife.
1. Approach. Most biochemical agents will be applied in a manner
that will result in exposure to terrestrial wildlife. Even bio-
chemical agents that are identical to naturally occurring semio-
chemicals and hormones, when applied as pesticides, result in
exposure to terrestrial wildlife which would be different (e.g.,
in terms of route/ time, and/or amount) from that which would
occur under natural conditions. The Agency, however, realises
that most biochemical agents are applied at very low rates, and
also, many possess phyBiochemical properties (e.g., high volatility)
that reduce exposure to terrestrial wildlife. On this basis (i.e.,
low potential for significant exposure) the Agency permits a reduc-
tion in both number of tests and number of organisms per test
to support registration of biochemical agents. Testing guidelines
for biochemical agents are, therefore, reduced from those in Sub-
division t of the guidelines for conventional chemical pesticides.
Certain use patterns and formulations will greatly reduce
exposure of birds and mammals to biochemical agents (e.g., confined
traps, tabs nailed to trees, tree injections, and uses around
buildings). Biochemicals used in this manner would be confined to
very limited areas, unlike sprayed chemicals that contaminate a
variety of wildlife habitats when broadcast over wide areas.
Therefore, these use patterns and formulations could reduce expo-
sure of terrestrial animals to biochemical agents to a point where
further reductions in testing may be appropriate. The Agency
invites comment on specific use patterns and formulations which
would qualify for reduced testing.
2. Tier Progression* (a) Tier Z and progression to Tier II.
Under these guidelines, two tests on birds normally are indicated
for all biochemical pesticides that are not highly volatile: an
avian single-dose oral toxicity test (§ 154-6) and an avian die-
tary toxicity teat (f 154-7). (See § 154-1, Pigvre 1.1--Tor bio-
chemicals that are highly volatile, only an avian single dose oral
toxicity test is indicated. The Agency is following this approach
because it believes that dietary exposure of a highly volatile
biochemical agent would not provide data useful for hazard assess-
ment , sine* the* Bdd£hemical agent would bevsquicfcly lost from the
feed.
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Sections 154-6 and -7 will provide Adequate data on toxic
effect*, including abnormal behavioral effects, of biochemical
pesticides to avian wildlife* One control group and one treatment
group per test nay be all that are necessary to provide satisfac-
tory data showing no adverse effects* These tests are similar to
JJ 711 and -2 in Subdivision E of the guidelines.
Toxicity data for human safety, section series 152, in most
cases would be sufficent to assess potential effects to wild mam-
mals. Tier XI tests are indicated if any of the following occur:
- (1) Maximum estimated environmental concentration (maximum ZSC)
is greater than or equal to 1/5 the avian single-dose oral LD50
value converted to ppm or is greater t***" or equal to 1/5 the
avian dietary LCSO value;
- (2) Signs of abnormal behavior are observed in the avian single-
dose oral or toe avian dietary toxicity tests at levels equal to
or less than the maximum EEC;
- (3) Growth, development, or reproductive effects may be expected
based on observed effects in the avian dietary toxicity test,
available fate data, use pattern information, and results of
tests required to support human safety, section series 152.
If none of the criteria above is met, additional testing at higher
tiers (II through V) is ordinarily not indicated.
(b) Tier II and Progression to Tier III. No additional test-
ing at higher tiers would ordinarily be indicated if:
- (1) Environmental fate characteristics indicate that the esti-
mated environmental concentration of the biochemical pesticide
in the terrestrial environment is less than 1/5 the avian die-
tary LCSO or the avian LD50 converted to ppm; and
- (2) The pesticide or any of its metabolites or degradation
products are not stable in the environment and potentially toxic
amounts are not likely to persist in avian feed.
Testing as outlined in Subdivision S of the guidelines
would be the next step (Tier III, $ 154-12) if:
- (1) environmental fate characteristics indicate tftat estimated
concentration of the biochemical .pesticide .la .t||fe£mrre«trial
environment is equal to or greater than 1/5 the avian dietary LC50
or the avian singledose oral LD50 converted to ppm; or
- (2) The pesticide or any of its metabolites or degradation prod-
ucts are stable in the environment to the extent that potentially
toxic amounts may persist in avian feed.
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3. Major Issues. In the process of developing testing guidelines
for terrestrial animals, the Agency recognized at least two areas
that require outside input and cosnent. A discussion of these
issues follows.
(a) Maximum test concentrations and doses, The Agency believes
that satisfactory data can be generated from the avian single-dose
oral toxicity test (§ 154-6) and the avian dietary toxicity test
(§ 154-7) where only one concurrent control group and one treatment
group are tested, if a maximum test concentration or dose is tested.
Negative results from such tests would provide a high degree of
confidence that no adverse effects are likely to occur from the
actual use of the biochemical pesticide.. If, however, effects are
observed at these maximum test levels, then further testing at
lower levels would be indicated in order to establish precise LC50
and LD50 values and corresponding 95 percent confidence limits.
Since most chemical pesticides are applied at rates measured in
pounds active ingredient (AX) per acre {or in kilograms/hectare) , and
biochemical pesticides are usually applied at much lower rates [often
measured in grams (ounces) AI per acre] , an appropriate maximum con-
centration or dose should reflect the lower application rates.
Therefore, the Agency has reduced the "»^ r»™> testing concentration
(5000 ppm) and dose (2000 mg/kg) established in Subdivision E for
avian toxicity tests on chemical pesticides, The established levels
are multiplied by the ratio of the proposed application rate in grams
to the number of grams in one pound (454 g) . The general equations
are as follows:
maximum application rate
Maximum Teat Dose « in grams AI per acre* x 2000 mg/kg
454g**
application rate
Maximum Test „ in grams AI per acre* x 5000 ppm
Concentration 454 g**
For example, a 20 gram/acre (49 gram/hectare) rate would give an
88 mg/kg maximum test dose and a maximum testing concentration of
220 ppm.
(b) Categorization of semioehemicals by structure/activity
relationships. Categorization of chemicals is a method through which
scientists can infer which chemicals present risks of harm to humans
and to the environment (Slesin and «andl«rY 1978). •*wl'«««tegoriza-
tion scheme that may be useful for determining hazard to terrestial
• For biochemical pest control agents.
•• A typical application rate for conventional pesticides.
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animals from semiochemicals used as pesticide* (e.g., pheroaones) is
based on structure/activity relationships. The study of structure/
activity relationships seeks to find association between a sub-
stance's physical and chemical properties and its effect on biolog-
ical activity (Slesin and Sandier, 1978). Recently McLeese et
al., (1979) used structure/activity relationship in the assessment
of adverse effects of sane industrial chemicals to nontarget organ-
isms, such as shrimp and clams. Since most semiochemicals used as
pesticides are applied at very low rates and possess special physi-
cochemical properties (such as high volatility) that lessen their
exposure to terrestrial animals, acute toxlcity data on terrestrial
animals for each new semiochemical submitted for registration may
not be necessary. Rather, a determination of hazard could be based
on existing acute taxicity data for structurally similar semiochem-
cals.
These guidelines for biochemical pesticides do not include use
of structure/activity relationships. However, the Agency believes
that this concept, when developed for semiochemicals, could provide
aa acceptable data base for hazard assessments concerning both
terrestrial and aquatic animals. At the same time, it could reduce
the data required for registering semiochemicals used as pesticides.
A-3. Aquatic Animals.
1. Approach. Many biochemical agents possess special physicochem-
ical properties, or are applied in a manner such that they are
unlikely to enter the aquatic environment in significant quantities.
Consequently, they would not usually be expected to pose hazards
to aquatic animals. It is on this basis (i.e., low potential for
significant exposure) that the Agency reduces aquatic animal data
guidelines for certain biochemical agents from those for conven-
tional chemical pesticides in Subdivision E. Guidelines are
reduced in two ways:
- (1) The fish acute bioassay test indicates that only one fish
species, rather than two, be tested) and
• (2) One control group and one treatment group per test may be
all that are necessary to provide satisfactory data showing no
adverse effects.
Based on past experience with biochemical pe*tU014«s, the
Agency believes that most will qualify for reduced testing. But,
in any case, each biochemical screened out for reduced testing
would have several criteria supporting such a course of action.
They are:
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- (1) Non-aquatic use; and
- (2) Low potential for significant exposure, based on use pattern*
fo rmulation, and/or
- (3) Low use rates/ and/or
- (4) High volatility.
A discussion of these exposure criteria and their use follows•
A biochemical agent oust o»et the first criterion plus one or
more of the last three criteria in order to qualify for reduced
testing. A biochemical agent that meets only one of the last three
criteria (low exposure use pattern/formulation, low use rates, or
high volatility) might be considered a w*afc candidate for reduced
testing, while one that meets tvo or three criteria would be a ouch
stronger candidate.
(a) Non-aquatic use site. This criterion identifies those
biochemical agents that are applied on land when used as directed.
Biochemical agents that meet this criterion (are applied on land)
qualify for reduced testing. Aquatic animals will be exposed when a
biochemical is applied directly to water (e.g., mosquito larvicides
and aquatic herbicides that are biochemicals), and the Agency
believes that reduced testing for these biochemical agents is not
warranted.
(b) Low exposure via use pattern or formulation. Use patterns
or formulations such as confined traps/ tree injections, hollow
fibers, tape dispensers, and drip irrigation, greatly reduce pos-
sibility of aquatic exposure. Biochemical agents applied in one of
these manners would qualify for reduced testing. There may be
other use patterns or formulations that would largely preclude
aquatic exposure. Also, there are many borderline situations that
may warrant reduced testing, such as minor uses, single applications/
ground applications, or soil incorporation. Consultation with the
Agency may be necessary to evaluate these borderline situations on
an individual basis•
(c) Low use rate, the Agency provides low use rate as a
criterion for reduced testing on the premise that application of
small quantities of biochemical in a terrestrial use pattern limits
the amount of material available to reach water and reduces the like-
lihood that concentrations will be high enough to^'KaeK'a hazard
level. Biochemical agents applied on land at 0.7 ounces (20 grans)
active ingredient or less per acre per application would qualify
for reduced testing.
An alternative approach would be to define low rates in relation
to the biochemical's natural (ambient) concentration in the environ-
ment. For example, if application of the biochemical increases its
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41
concentration to no more than two tiaes the ambient concentration/
then the. rate used would be considered low and the biochemical •
would qualify for reduced testing.
(d) High volatility. High volatility is a physical character-
istic of some biochemicali that, for terrestrial use patterns,
would almost preclude potential for aquatic exposure. An insect
pheromone, whose efficacy often relies on vapor phase contact by
the target insect, is an example of a biochemical that meets this
criterion. For these guidelines, biochemical agents with an esti-
mated volatility (H) greater than 5x10"5 atm. mVmole would qualify
for reduced testing. Such biochemicals would have a volatilization
half-life of less than one day, and therefore would not persist in
water.
2. Tier progression. Biochemical pesticides determined to have
low potential for aquatic exposure would qualify for reduced test-
ing but would still be required to have one freshwater fish acute
bioassay (§ 154-8) and one aquatic Invertebrate acute bioassay
(§ 154-9) to support registration (see J 154-1, Figure 2). Pro-
tocols for these tests are similar to §§ 72-1 and -2 of Subdivi-
sion E. The Agency recognizes that these tests are designed to
assess acute toxicity, and may not be entirely suitable when other
modes of action are concerned. Nevertheless, the Agency believes
that, until a more appropriate screening test is available, these
bioassays, in combination with the screening criteria for reduced
testing, provide adequate evidence as to whether aquatic exposure,
if any, will be biologically significant.
Tier IX Environmental fate tests (§§ 155-4 through -13) would
be indicated if any one or more of the following occur:
- (1) Signs of abnormal behavior are observed in Tier I tests at
concentrations equal to or less than the maximum expected
concentration in water; or
- (2) The maximum expected concentration in water is equal to or
greater than 0.1 of any EC£Q or LCso determined in testing
required by §J 154-8 or -9; or
- (3) Maximum expected concentration in water is equal to or
greater than 0.01 of any BC50 or LC50 determined in testing
required by §5 154-8 or -9; and
- (4) Adverse effects on growth, denreloysMntr «r jap*eduction may
be expected based on Tier I test data, available fate data (e.g.,
from the product's research and development), use pattern
information, or available effects data on phylogenetically
similar target species.
If r.one of the above criteria are met, then additional testing at
higher tiers is not indicated.
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42
Biochemical agents that have terrestrial use patterns but do
not Beet any of the other three criteria for low exposure potential
may still not pose a significant hazard to aquatic organisms. But,
at present, there are not sufficient data on the effects of bio-
chemical pesticides to warrant fully reduced testing for these
materials. Instead, an intermediate course of testing is provided,
including both Tier I (aquatic animal tests, §§ 154-8 and -9) and
Tier XI (environmental fate tests, H 155-4 through -15} to support
registration. Results of Tier I and Tier II tests would be eval-
uated, and any further testing, if needed, would proceed along the
tier system in Subdivision E of the guidelines.
Biochemical pesticides applied to water do not qualify for
reduced testing because exposure of nontarget aquatic fauna is
unavoidable in such situations. The effects tests specified by
the tier testing system in Subdivision E (§§ 72-1 through -6) and
environmental fate testing (Tier II, Subdivision M) to support
registration apply to these pesticides. Progression to Tier II
testing applies only to biochemical agents screened out for reduced
testing at Tier I, since Tier II testing automatically applies
for all other biochemical agents. The data for Tier II are des-
cribed in the Environmental Fate sections (§§ 155-4 through 13) of
these guidelines.
If environmental fate characteristics indicate that the
estimated environmental concentration of the biochemical agent in
the aquatic environment is equal to or greater than 0.01 of any
EC50 or LC£o determined in testing in §5 154-8 or -9, then
testing as in Subdivision E of the guidelines would be indicated
(Tier III, § 154-13). If the estimated environmental concentra-
tion is less than 0.01 of the above-described toxicity values,
then no additional testing would be indicated.
3. Major Issue. Refer to part 711 A-2.3.b. of this Discussion for
a discussion that also applies to aquatic animals: categorization
of semiochemicals by structure/activity relationships.
A-4'. Nontarget Plants.
1. Approach. The plant testing scheme is based on the tier testing
scheme found in Subdivision J of the registration guidelines (Hazard
Evaluation: Nontarget Plants). Those guidelines andtheijr accompany-
ing discussion should be perused with respttot'to tBf^Psflt*ti.«r
testing scheme, the tests, the dose and other testing information
and the tier progression criteria. Testing procedures for biochem-
ical agents should be similar to those for other chemical pesticides
vith respect to phytotoxicity studies, and would therefore be sub-
ject to the same guidelines of Subdivision J.
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2. Tier Progression. Progression to Ti«r III and further plant
effects testing would depend on whether there are any adverse
effects to desirable plants at the Tier I level, and whether there
is possible movement by soil, water, or air from the intended site
of application to nontarget areas as determined by selected tests
of Tier II (Environmental Pate, Series 155; see § 154-1, Figure 3).
In the vast majority of biochemical pesticides, such movement might
occur, but at levels far below those which would have a detrimental
effect as determined in the Tier I tests.
A-5. Nontarget Insects*
1. Terrestrial insects, (a) Approach. Development of baseline
(first tier) tests for biochemical pesticides is difficult, for the
following two reasons:
- (1) Effects of these biochemicals will often be long-term (e.g.,
effects on growth) rather than acute; this type of activity does
not lend itself to short term testing; and
- (2) Effects on development or behavior, unlike mortality, may
be difficult to quantify.
Due to these factors, and due to the fact that research in
this area is in the early stages of development, there are no widely
accepted, simple tests for evaluating biochemical effects on behavior
and development of nontarget insects.
In view of the above, the Agency will not outline any specific
type of Tier I testing for'effects of biochemical pesticides on
nontarget insects. Rather, the registrant should report any adverse
effects on nontarget insects noted during efficacy testing, includ-
ing effects such as:
- (1) Mortality or other adverse effects (e.g., behavorial
modification) on insect predators or parasites of the target
organism; and
- (2) Direct adverse effects on pollinators, or repellent effects
on pollinators.
If no such effects are noted during efficacy testing, and in
the absence of any other data indicating potential for adverse
effects, no nontarget insect testing will be indicated. However, if
adverse effects are noted and/or auxiliary data indicate a potential
for adverse effects, then Tier II (Environmental Fate) testing will
apply. If fate data do not indicate exposure, no further testing
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44
will b« indicated. If fate testing indicates exposure, the registrant
should consult with the Agency regarding further testing. Testing
at that point would most likely be •isolated or actual field test-
ing, and would be directed at the problem identified during efficacy
testing or indicated by the auxiliary data.
(b) Tier progression. (1) Tier I. Ac pointed out in the
preceding discussion on "Approach", the Agency will not develop any
baseline data requirements specifically to assess effects of bio-
chemical pesticide* on nontarget terrestrial insects. Rather,
Tier I vill consist of data (such as efficacy data), submitted by
the registrant or made available from another source. If no data
exist to indicate potential for adverse effects on nontaxget insects,
no testing will apply. (See f 154-1, Figures 4 and 5.)
(2) Tier II. The data for Tier II »re described in environ-
mental fate testing, section series 155 of these guidelines. Should
environmental fate testing indicate no potential for nontarget
insect exposure, no further testing would be indicated. Indication
of exposure potential would lead to further testing.
(3) Tier III. The registrant will have to consult the Agency
prior to testing at the Tier III level. Testing required at this
point would most likely be simulated or actual field testing; the
specific type of testing required will depend on the type of problem
identified during efficacy testing or indicated by auxiliary data.
2. Aquatic Insects, (a) Approach. Testing should follow the
scheme outlined in Aquatic Animal Testing Scheme for Biochemical
Pesticides at § 154-9. Results of testing through this scheme vill
answer:
- (1) Does aquatic invertebrate bioassay indicate potential
adverse effects?
- (2) Does environmental fate testing indicate potential for
aquatic exposure?
If the answer to either of these questions is "no," no further
testing is indicated. If the answer to both is "yes," then testing
should continue according to Subdivisions S and X>.
(b) Tier progression. A* noted above, no specific tests
have been developed exclusively for assessing the haxards^of bio-
chemical pesticide* to nontarget aquatic insects, father/ the
tier "system" used here is constructed from testing systens
developed in other parts of the registration guidelines. Testing
should be conducted according to §154-9 of Ap^atic Animal Tier
Testing Scheme for Biochemical Pesticides, If bioassays indicate
potential adverse effects on nontarget injects and Tier II (en-
vironmental fate) data indicate potential for aquatic exposure,
testing should be conducted according to Subdivisions E and L.
If one of these potentials is lacking, then no further testing will
be indicated.
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45
B. Mierobial Pest Control Agent*.
B-1. Terrestrial Wildlife and Aquatic Animals (Central)
1. Approach. In designing Tier 1 teats, the Agency has attempted
to balance two opposing philosophies concerning the hazards of
microbial pest control agents4 On th« on« hand, due to the rela-
tively small existing' data base pertaining to aicrobial pest con-
trol agents and their theoretical potential for causing environ-
mental damage, the Agency would wish to conduct extensive testing,
regardless of the pesticide's use pattern. This would be done in
an effort to determine, as conclusively as possible, whether or
not the pesticide's host spectrxn includes nontarget terrestrial
and aquatic organisms. The opposing philosophy argues that exten-
sive testing in search of nontarget hosts is not justified because:
- (1) The pesticide organisms have existed in nature for thousands
or millions of years without affecting the nontarget organisms with
which we are concerned;
- (2) Rather extensive testing on a few microbial pest control
agents (e.g., Bacillus thuringiensis and the nuclear polyhedrosis
viruses) has demonstrated the safety of these types of pesticides;
- (3) The application of a microorganism does not necessarily
increase that particular aicrobial population to a level any
higher than would have occurred under natural conditions; and
- (4) The known hazards of the classical chemical pesticides are
far greater than any hazards known to exist vith microbial pest
control agents.
After considering the two aforementioned philosophies, the
Agency has concluded that at least some (minimum) test data on
terrestrial and aquatic organisms should usually be evaluated,
regardless of the pesticide's site of application (outdoor) and
apparent potential for exposure. These minimum data would be
necessary for the following reasons:
(1) Nhen a microbe is applied as a pesticide, great numbers
of the microbes are placed in the environment outside (apart from)
its host, at a discrete point in time (day of application), and
spread over all living and nonliving components of the target site,
as well as adjacent areas (due to drift)» hence, in terns of numbers
of nontarget organisms exposed, the number of different species ex-
posed, and the degree of exposure (number of microbes per nontarget
organism), exposure would probably be greater than under natural
conditions; and
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46
(2) Data on toxic or pathogenic effects are essential for haz-
ard assessment purposes when terrestrial or aquatic organism are very
likely to be exposed to a microbial pest control agent, especially
when no fate data will be required by the Agency in the first tier of
testing.
Pathogenicity and toxicity are the major effects of concern
regarding terrestrial and aquatic organisms. Therefore, the Agency
has developed guidelines that will allow hazard assessment of
possible pathogenicity and toxicity problems. In addition, the
Agency desires a high level of confidence that no adverse environ-
mental effects will result from actual use of microbial pest con-
trol agents. Toward this end, the guidelines in Tier I reflect a
maximum hazard approach to testing as described earlier. Negative
results from tests using this approach would provide a high degree
of confidence that no adverse effects are likely to occur from the
actual use of microbial pest control agents. Prior to the Agency's
considering the registration of naturally occurring and strain-
improved microbial agents, applicants would submit only Tier I
data on nontarget organisms. However, both nontarget organism
data (Tier I) and environmental expression data (Tier XI) would be
evaluated prior to considering genetically engineered microbial
pest control agents for registration.
2. Major issues, (a) Maximum hazard dosage levels. Unlike
environmental levels which generally decrease following application
of chemical pesticides, the environmental levels of microbial p«st
control agents 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 y«»<»m« amount of active ingredient (microbial agent 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 b* available to nont&rg«t terrestrial wild-
life and aquatic animals following a pesticide application. The
maximum amount of microbial pest control agent (active ingredient)
that one infected host can contain is called the host equivalent in
these guidelines. Since the host insect can vary greatly in size,
and the number of hosts that could be consumed by a known terrestrial
or aquatic predator will also vary, largely d»p«nd4A!i upon its size,
the host equivalent was adjusted by a weight-to-weigW ratio of test
animal to infected host organism. Therefore, the maxiaum hazard dose
in these guidelines is equal to the host equivalent multiplied by the
ratio of the weight of the test animal (e.g., fish or bird) to the
weight of the infected host organism, (e.g., insect larva*) The
route(s) of administration (e.g., oral, pazentexal) and the size
of the test organism(s) will largely determine whether the maximum
hazard dose will be a multiple or a fraction of the adjusted host
equivalent amount. Obviously, the maximum hazard dose must be an
amount that is technically feasible to administer to the test organ-
ism! s) and therefore the Agency will be flexible in its assessment
of whether a high enough dose was used in any given test.
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47
In eh* case of microbial pest control agents applied to the
soil to .control soilbornc diseases affecting plants, the soil may
contain the highest concentration of the agent. The host equivalent
concept would not be applicable in this situation since the host,
most often, will be a microscopic propagule (spore, ooepore,
sclerotium or chlaoydospore ) .
The Agency realises that it would be very difficult to estab-
lish specific IC^Q, XDso' or L°50 values (e.g., LOSO - 1000 mg/kg)
and 95 percent confidence limits for aost microbial pest control
agents, because test data are not likely to exhibit a log-probit
dose-response relationship that is typical of chemical pesticides.
Therefore, data that establishes that the LCso» *DSO • or 3-°50 im
greater than the maximum hazard dosage level (e.g., LD50 >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.
(b) jtMviimnn hazard routes of administration. Various routes
of administration are provided in these guidelines. There is a
general belief in the Agency, however, that a parenteral route
[e.g., intravenous (IV), intraperitoneal (IP)] would provide an
acceptable maximum hazard exposure to terrestrial and aquatic
animals in Tier I tests. While this route of administration is
environmentally unrealistic, the Agency believes that negative test
results from testing by IV or IP injection and using maximum hazard
dosage levels would provide a high level of confidence that no
adverse effects would occur from the actual use of the microbial
pest control agent.
(c) Age of the test animals. The Agency considers that suf-
ficient iamunological and physiological differences exist between
Immature animals and mature animals to suggest that immature animals
are potentially more susceptible to infection and possibly to the
effects of any toxin produced by the microbial pest control agent.
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 mmv •»«"•"« hazard approach to testing.
(d) Methods for detecting effects. Unlike toxicity tests
where mortality can usually be determined by observation, infac-
tivity tests often require sophisticated assessment methods for
detecting sublethal pathogenic effects. Due to the extremely
diverse nature of the active component in microbial pest control
agents [e.g., natural toxins, acellular agents (viruses), prokary-
otic cells (bacteria), eukaryotic cells (fungi, protozoans, most
algae)], many different methods to detect each agent and assess
infections have been developed. To assist applicants and regis-
trants, the Agency is providing a scheme which will show some
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48
acceptable methods for detecting pathogenic effects for each type
of aicrobial pest control agent (Table 1). This scheme is a gen-
eralization and is not completely reliable. However, it should
cover a majority of the microbial agents and methods for detection.
(e) Viruses; the most challenging problems. Viruses represent
the most challenging problems because they are parasites at the gene-
tic level. Unlike other pathogens, they are acellular in their para-
sitic state and are able to insert their genomes (genes) directly
into the host cell(s) with no intervening parasite membrane (Kawanishi,
1979). Very often , the diseases caused by viruses are preceded by
long periods of latency. Viruses in latent states are often unde-
tectable by microscopy or serological techniques. Some of the
detection methods proposed above could detect viruses even when
they are cell-associated in the form of nucleic acid molecules.
The Agency is not aware of acceptable screening test(s) to determine
if viruses in latent states will actually cause adverse effects
later on. Further research is needed in this area.
Detection of a virus in a latent state ordinarily would b«
considered a nonin-fectious effect. Noninfectious effects have been
reported in infactivity tests with other microorganisms. For
example, Ignoffo (1973) reports that noninfectious bacteremia and
the presence of bacteria in tissue of vertebrates following admin-
istration of heavy doses of Bacillus moritai and B. thuringiensis
have been observed. At the present tine, the Agency has decided
to place infectivity as its primary concern. For the present, the
decision to require effects, excluding toxicity, will be handled
on a case-by-case basis.
(f) Test protocols. Mo standard widely accepted test proto-
cols are available to evaluate the safety of microbial pest control
agents to terrestrial and aquatic animals. In lieu of established
test protocols, the Agency is providing tentative guidelines,
based on past experience and on selected published references,
which would aid in the development of generally accepted testing
standards and protocols.
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49
Table i—METHODS FOR DETECTING EfKKCTb OF MXCROBIAL PEST COXTROL AGENTS IN
SAFXXY TESTS
Hicrobial y««t Control Agents
Method of
Assessment tticrobial Toxin
Bistopathology 0
Serology* N.A.
Nucleic Acid K.A.
Hybridization**
Cellular
JuS'tt&^S
Fungi,
Bacteria)
X
0
B.A.
arell tiler
Agent
(virus;
X
X
0
Key to Table symbol*:
0: All members of this group detectable by this method
X: Not all infections by this group are detectable by this method
S.A.: Not Applicable
•Sadioiomunoassay, Enzyme-linked Imrnunosoxbent Assay,
laarunoperoxidase Assay, Immunofluorescence Assay.
**OKA:DftA and KHA:T7RA HybridLzation Techniques.
(Source; C.Y. Xa*«nishi, 1979)
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50
B-2. Terrestrial Wildlife.
1. Approach. These guideline* indicate two tests on bird* for
all microbi&l pest control agents: an avian single-dose oral
toxicity and pathogenicity test ({ 154-16) and an avian injection
pathogenicity test (§ 154-17). The avian single-dose oral toxiclty
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 oral exposure. The duration of the
study would be about 30 days in order to provide tiae for incuba-
tion/ infection, and manifestation of pathogenic effects from the
microorganism. Also, gross necropsies are indicated, and any
lesions would have to be characterized. This test is a modifica-
tion of the avian single-dose oral U>so study found at § 71-1 in
Subdivision £ of the guidelines.
The avian injection pathogenicity test would provide data on
the pathogenic effects of the mlcrobial pest control agent on birds
following a parenteral exposure. The guidelines for the duration
of the test and gross necropsies are similar to the avian single-
dose oral toxicity and pathogenicity test. In addition, however,
Investigation of specific organs, organ systems, and the site of
injection for multiplication of the mlcrobial pest control agent,
are indicated. Further, any observed pathogenic effects (e.g.,
lesions) lead to an assessment of cause and a description of the
detection methods used in the assessment. A standard protocol for
this test is not currently available, although Friend and Trainer
(1974 a and b) have administered duck hepatitis to mallard ducks
via two injection routes: intravenously and intraperitoneally.
One combined test would necessitate the administration of the
mlcrobial pest control agent via two different routes (e.g., oral
and intravenous injection) to the same group of test birds. A
standard protocol for this combined test is not currently available.
The two routes of administration would expose the mlcrobial pest
control agent to two radically different environments in the bird:
the gut and the blood.
2. Tier Progression, (a) Tier I. If no toxic or pathogenic
effects are observed after exposing birds to the mlcrobial pest
control agent via two different routes of administration (oral and
injection) at the «*yi«^n hazard dosage levels/ then ao farther
testing of birds would be indicated. If toxic or pathogenic effects
are observed at the maximum hazard dosage levels, then Tier IX
environmental expression tests ($$ 155-18 through -20) would be
indicated. (See $ 154-1, Figure 6.)
Data on wild mammal toxicity and pathogenicity ($ 154-18) are
indicated on a case-by-case basis when data indicate that there is
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51
considerable variation in the sensitivity of different mammalian
species to the effects of a mierobial pest control agent, and when
wild mammals would be heavily exposed to the microbial pest control
agent under normal use. However, the toxicity and pathogenicity
data in section series 152 of this Subdivision for evaluating
hazard to humans and domestic """•If are normally adequate to
indicate hazard to wild mammals. If no toxic or pathogenic effects
are observed in tests on manuals, then no further testing of wild
maomals would be follow, if any effects are observed in tests on
wild mammals, then Tier II environmental expression testing (§§ 155-
16 through 155-20) would be indicated.
Genetically engineered mierobial pest control agents are
treated differently from mierobial pest control agents that are
identical to naturally occurring microorganisms or that are improved
strains of naturally occurring microorganisms. Since genetically
engineered microorganisms do not pre-exist in the environment, data
on effects on nontarget birds and mammals in Tier I ($§ 154-16
through -18) and data on survival in the environment in Tier II
(H 155-18 through -20) are indicated in order to assess potential
environmental hazards.
(b) Tier IZ. The data outlined in Tier II are described in
environmental expression testing (§§ 155-18 through -20) of these
guidelines. If the expression characteristics preclude exposure of
the mierobial pest control agent to nontarget birds and mammals,
then no further testing of these animala would be indicated. If
Tier II tests indicate that birds and mammals will be exposed to
themicrobial pest control agent, then testing at Tier III would
follow.
(c) Tier III. The types of effects reported in the Tier I
tests would determine which Tier ill test(s) would apply. If toxic
effects are reported in Tier I tests, and Tier II tests indicate
exposure, then the guidelines of $ 71-2 of Subdivision E would
apply (see J 154-24). In this case, futher testing (if needed)
would proceed as in Subdivision E. If pathogenic effects are
reported in Tier I tests at an amount equal to the adjusted host
equivalent, or if chronic, carcinogenic or teratogenie effects are
reported in tests in §§ 152-50, -51, and -53 for evaluating hazards
to humans and domestic animals, then a long-term avian pathogenicity
and reproduction test (§ 154-26) would apply. This test would pro-
vide data on pathogenic effects of the mierobial pest control
agent on birds during a sensitive period in their- life, breeding
aad reproduction. It would also providt^dats oa £he effects of the
mierobial pest control agent on avian reproduction. This test
would be a aodification of the avian reproduction study (§ 71-4) in
Subdivision E of the guidelines. 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.
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52
Pathogenic effects occurring at Tier III And beyond raise
serious questions concerning the registration of any microbial pest
control agent. Also, testing at Tier XV, simulated and actual
field testing for mammals and birds (J 154-33) nay not be feasible/
since it may not be possible to confine the microbial pest control
agent to a test area and prevent it from escaping to contaminate
adjacent areas. The applicant should seriously reconsider the
proposed registration of any microbial pest control agent that
requires Tier ZZI or IV testing* If a decision cannot be made
without further testing/ and the microbial pest control agent can
be restricted to a field test area, then testing at Tier IV
would be indicated.
(d) Tier IV. Simulated and actual field testing ($ 154-33)
would provide data on the pathogenic effects of the microbial pest
control agent on birds and masnals following field applications at
actual label use rates. This test would be indicated when pathogenic
effects are reported in Tier III testing (§ 154-26) at levels equal
to actual or expected field residue exposure levels, and when the
Agency is reasonably confident that quarantine methods will prevent
the microbial pest control agent froa escaping from the test area
to contaminate adjacent areas. The specific type of test (small-
pen, large-pen, or full-scale field test) should be discussed with
the Agency before beginning the study.
3. Major issues for discussion. In the process of developing the
guidelines for terrestrial animals, the Agency recognized many im-
portant areas that require outside input and comment. The Agency
needs scientific input and invites comments on the following issues
(a) in vivo testing. The guidelines outline in vivo testing
of birds and mammals. _l£ vitro testing may be considered in the
future. Wolf (1975) has suggested a two-pronged testing approach
for safety testing of baculoviruses, using both ^n vivo and tissue
culture (TC) testing. He reported that there are established or
permanent cell lines for duck embryo fibroblasts, chicken embryo
fibroblasts, as well as 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 b««n tested
in vitro in either aviaa 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 vfcea -vabbits aad**to« were injected
or fed the latter virus.
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.
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53
(b) Test substance. Microorganisms-used as pesticides could
be applied in any on* 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 microbial agent in which the toxin would be most readily avail-
able. Unfortunately, there is no easy way to determine which is
the most infectious or toxic form of the microorganism 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 polyhedryde,
respectively.
For these guidelines, testing the technical grade of the active
ingredient applies in all tests except the simulated and actual field
testing {$ 154-33), when the use of the formulated product applies
in order to simulate or reproduce actual field-use. This provides the
consistency between the tier tests necessary for assessment of hazard.
(c) Route of administration. These guidelines outline testing
by oral gavage and via injection by a parenteral route (preferably
intravenous or intraperitoneal). It is important to note that the
administration of test material to 10 to 17 day old birds by oral
gavage will likely require the use of small needles or cannulae with
ball-tipped ends in order to prevent injury to the birds. Ignoffo
(1973) reported that the following groups of terrestrial animals
have been tested in vivo for effects caused by entonopathogens:
Group Routes of Administration
Marauds (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)
At the present time there is no general consensus within the
Agency concerning the route of administration that would consis
tently provide the maximum hazard exposure to nontarget birds or
mammals. There is, however, a general belief that one of the
parenteral injection routes may be appropriate. Also, since the
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54
gut normally provide* such a radically different environaent from
that in the rest of the bird or mammal body, and since insectivorous
birds and mammals can be expected to ingest large quantities of
actively growing microorganisms when they feed on diseased insects,
the Agency believes that the oral route would also be appropriate.
The dietary route of administration was considered for Tier Z
tests, but the Agency beieves that it does not generally reflect
the Baxioua hazard test philosophy for Tier X tests. The diet,
however, is considered to be an appropriate route of administration
for Tier IZZ and Tier XV tests (§§ 154-26 and 154-33).
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 manuals. Xf any such effects
were to be reported in safety tests on domestic ruminants, then
the Agency would solicit similar tests on wild ruminants.
The Agency recognizes that a combination of administrations in
one test (e.g., oral and intravenous or intraperitoneal injection)
may be possible. Xt 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 organisms so as to cause mortality, or in some other way cause
spurious results.
(d) Avian test species. These guidelines provide that young
bobwhite quail or mallard ducks be tested in Tier X tests. Birds
between 10 and 17 days of age at the beginning of the test period
should be used in the avian single-dose oral toxicity and pathogen-
ic ity test and in the avian injection pathogenicity test. Within
a given test, all birds should be the same age. (U.S. Environmental
Protection Agency, 1978).
Suoners 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.
Xn 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 docks in avian toxicity and >*thogenicity
tests of microbial pest control agents:
- (1) These species are ecologically significant and widely
distributed in the United States;
- (2) They have proven to be good laboratory test species and are
appropriate for acute, subaeute, and chronic testing;
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55
- (3) Laboratory populations are genotypically and phenotypically
comparable to wild species;
- (4) There is a large body of data on the effects of chemical
pesticides on these species available for comparison purposes;
- (5) It ha* 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
- (6) Testing species from the family Icteridae (e.g., blackbirds,
gracklss, 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 two
or three 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 pathogenieity testing/ the Agency feels that
sufficient imsunological and physiological differences exist between
immature birds and adult birds to warrant considering the immature
bird as potentially more susceptible to infective challenge, and so
proposes their use in the maximim hazard testing approach.
(e) Selection of dose levels. For Tier I tests, the Agency
suggests that a aaxl man hazard dosage be administered. For the
acute oral test for toxicity and pathogenieity, the maximum hazard
acute oral dose should be no less than 10x the adjusted host
equivalent amount. [The adjusted host equivalent is equal to the
host equivalent times the ratio of the weight of the test aniaals
to the weight of the host organism. The host equivalent is equal
to the maximum amount of active ingredient that one infected host
(e.g«, a late instar larva) can contain.] If the calculated amount
of microorganism is determined to be excessive, a Sx or 2x amount
may be used. If the microbial pest control agent produces any
toxic or pathogenic effects at the •"!*•'**viff hazard dose level, then
testing at lower doses would be indicated* If the microbial agent
kills test organisms at the the highest dose level, then sufficient
doses and test organisms would be required to determine an LD50
value, if possible. For the injection pathogenieity tests, the max-
imum hazard dose level should not be less than one adjusted host
equivalent. If this amount is not feasible, a j/3jfoj Vs, *' or 1/1 Ox
amount could be tested if a rationale is proviiSdHS irapport the
reduction. If the microbial pest control agent produces any path-
ogenic effects at the highest dose level tested, then testing at
lower doses would be indicated.
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56
It has been suggested (Ignoffo, 1973, and Summers, 1975) that
microbiological pesticides be tested at 10x to 100x the average
field dose per acre with a conversion ratio of the weight of the
test animals to the weight of a 70-kilogram nan. This may be appro-
priate for safety testa required for human safety, but the Agency
believes a different approach may need to be taken for safety
tests to provide data on effects to wild birds and mammals. The
Agency has information that one infected host organism ran contain
a quantity of active infectious agent greater than the amount
applied to one acre. Considering that diseased host insects are
likely to lose their natural defense mechanisms and become easy
prey for birds and mammals, and that birds and mammals can consume
more than one infected host in a day, a *""f1npir Hazard dosage should
be greater than the amount of microbial pest control agent in one
infected host. Since the number of infected hosts that a bird or
maanal can consume is a factor of the size of the predator and the
size of the infected host, the host equivalent can be adjusted by
the factor equal to the ratio of the weight equivalent can be adjusted
by the. factor equal to the ratio of the weight of the test animal to
the weight of the host organism:
adjusted host . ho-t €
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57
(g) Synergism with chemical pesticides. The Agency 1* aware
of the synergistic effects of some chemical pesticides. Where
information is available, the Agency uses it to determine potential
hazards from combinations of chemical pesticides in formulations
and from sequential applications of different chemical pesticides.
At the present time, the Agency does not regularly require applicants
to test for synergistic effects from chemical pesticide combinations.
Successful pest control has been achieved- with some combinations
of microbiological and chemical pesticides. They offer the benefits
of ionediate pest reduction plus long-term control. Friend and
Trainer (1974 a and b) reported findings that suggest synergistic
response in mallard docks to the combined effects of different
organochlorine pesticides and duck hepatitis virus (DHV) when com-
pared to the effects of the different organochlorine pesticides
(DDT and Dieldrin) and DHV alone. A similar interaction could
potentially occur with microbial pest control agents that exhibit
effects in Tier X testing and chemical pesticides.
The Agency believes it would be inappropriate to impose a Tier
I requirement to test for synergism on microbiological pesticides
at this tine. However, if data suggest that a microbial pest control
agent and a chemical pesticide will act synergistically, and the
microbial pest control agent and the chemical pesticide will be com-
bined in one product for use outdoors, or if the labeling of the
microbial agent recommends combination use with the chemical pesti-
cide, then the Agency may request Tier I tests on the formulated
product in addition to Tier I tests on the technical grade of each
active ingredient. For all such combinations, the Agency will deter-
mine the need for additional data on a case-by-case basis.
(h) Synergism with other biorational pesticides. Combinations
of different microbial pest control agents as well as combinations
of a microbial pest control agent and a biochemical pesticide (e.g./
pheromone) are being tested for use in a single formulated product.
Preliminary tests have shown, in some cases, additive and synergistic
effects on the target organism(s). Although the Agency knows of no
instances where these combinations have resulted in additive or
synergistic effects on nontarget organisms, such effects are a
possibility. As with the previous issue [(g) above], the Agency
feels that it would be inappropriate to impose a requirement to
test for synergism on microbial pesticides at this time. However,
if any data suggest that a particular biorational pesticide, when
combined in a product for use outdoors, will act synergistically,
then the Agency may request Tier I tests on the formulated product
in addition to Tier I tests on the technical grade of the active
ingredient.
(i) Protocols. Generally acceptable testing protocols are
needed to complete and finalize these guidelines. In the interim,
scientifically sound protocols are acceptable subject to prior
review by the Agency.
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57
(9> Synergism with chemical pesticides. The Agency is aware
of the synergistic effects of some chemical pesticides. Where
information is available, the Agency uses it to determine potential
hazards from combinations of chemical pesticides in formulations
and from sequential applications of different chemical pesticides.
At the present time, the Agency does not regularly require applicants
to test for synergistic effects from chemical pesticide combinations.
Successful pest control has been achieved-with some combinations
of microbiological and chemical pesticides. They offer the benefits
of ionediate pest reduction plus long-term control. Friend and
Trainer (1974 a and b) reported findings that suggest synergistic
response in mallard ducks to the combined effects of different
organochlorine pesticides and duck hepatitis virus (DHV) when com-
pared to the effects of the different organochlorine pesticides
(DOT and Oieldrin) and DHV alone. A similar interaction could
potentially occur with microbial pest control agents that exhibit
effects in Tier I testing and chemical pesticides.
The Agency believes it would be inappropriate to impose a Tier
I requirement to test for synergism on microbiological pesticides
at this time. However, if data suggest that a microbial pest control
agent and a chemical pesticide will act synergistically, and the
microbial pest control agent and the chemical pesticide will be com-
bined in one product for use outdoors, or if the labeling of the
microbial agent recommends combination use with the chemical pesti-
cide, then the Agency may request Tier I tests on the formulated
produce in addition to Tier I tests on the technical grade of each
active ingredient. For all such combinations, the Agency will deter-
mine the need for additional data on a case-by-case basis.
(h) Synergism with other biorational pesticides. Combinations
of different microbial pest control agents as well as combinations
of a aicrobial pest control agent and a biochemical pesticide (e.g.,
pheromone} are being tested for use in a single formulated product.
Preliminary tests have shown, in some cases, additive and synergistic
effects on the target organism(s). Although the Agency knows of no
instances where these combinations have resulted in additive or
synergistic effects on nontarget organisms, such effects are a
possibility. As with the previous issue t(g) above], the Agency
feels that it would be inappropriate to impose a requirement to
test for synergism on microbial pesticides at this time* However,
if any data suggest that a particular biorational pesticide, when
combined in a product for use outdoors, will act synergistically,
then the Agency may request Tier X tests on the formulated product
in addition to Tier X tests on the technical grade of the active
ingredient.
(i) Protocols. Generally acceptable testing protocols are
needed to complete and finalise these guidelines. In the interim,
scientifically sound protocols are acceptable subject to prior
review by the Agency.
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58
Literature Cited
(1) Friend, M., end 0.0. Trainer. 1974a. Experimental DDT-
Duck hepatitis virus interaction studies. J. Wildl. Manage.
38(4):887-895.
(2) and . 1974b. Experimental
DieldrinDuck hepatitis virus interaction studies. J. Wildl. Manage.
38(4):896-902.
(3) Penner, P., B.R. McAuslon, C.A. Him*, J. Sanbrook, and
D.o. White. 1974. The Biology of Animal Viruses. Second Edition.
Academic Press, N.Y.
(4) tgaoffo, C.M., C. Garcia, R.W. Kapp, and W.B. Coate.
1975. An Evaluation of the Risks to Mammals of the Use of an
Xntomopathogenic Fungus, Noauraea rileyi/ as a Microbial Insecti-
cide. Pp. 354-359 in Baculoviruses for Insect Pest Control.
Safety Considerations. Selected papers from EPA-OSDA Working
Symposium American Society of Microbiology. Washington/ D.C.
(5) Ignoffo, C.M. 1973. Effects of entomopathogens on
vertebrates. Annals N.Y. Acad. of Sci. 217:141-164.
(6) Kawanishi, C.Y. 1979. Personal Communication. EPA,
HERL (MD 82) Research Triangle Park, North Carolina. 27711.
(7) McLeese, D.w,, v. Zitko, and M. Peterson. 1979.
Structurelethality relationships for phenols, anilines and other
aromatic compounds in shrimp and clams. Chemosphere 2:53-57.
(8) Nordlund, D.A., and W.J. Lewis. 1976. Terminology of
chemical releasing stimuli in intraspecific and interspecific
interactions. Chem. Eeol. 2(2);211-220.
(9) U.S. Environmental Protection Agency. 1978. Registration
of Pesticides in the United States: Proposed Guidelines, Subpart E
-Hazard Evaluation: Wildlife and Aquatic Organisms, 1978 (July 10).
Fed. Reg. 43(132):29724-29737.
(10) Slesin, L., and R. Sandier. 1978. Categorization of
chemicals under the Toxic Substances Control Act. Kcol. Law Quart.
7:359-396.
(11) Summers, M., R. Ingler, L.A. Falcon, and *. Vail, eds.
1975. Guidelines for Safety Testing of Baculoviruses. Pp. 179-184
Ln Baculoviruses for Insect Pest Control: Safety Considerations.
Selected Papers from EPA-OSDA Working Symposium, American Society
for Microbiology. Washington, D.C.
(12) Wolf, K. 1975. Evaluation of the Exposure of Pish and
Wildlife to Nuclear Polyhedrosis and Granulosis Viruses. Pp. 109-
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59
111 in Baculoviruses for Insect Pest Control: Safety Considerations.
Selected Papers froa EPA-USDA Working Symposium, American Society
for Microbiology. Washington, D.C.
(13) Weiss, B. and V.G. Laties. 1979. Assays for Behavioral
Toxicity: A Strategy for the Environmental Protection Agency. Pp.
213215 in Test Methods for Definition of Effects of Toxic Substances
on Behavior and Neuromotor Function, Neuro-Behavioral Toxicology;
Vol.1. Suppl. 1. ANXHO International Inc. Report No. XPA 560/11-
79-010.
B-3. Aquatic Animals.
1. 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. They are:
- (1) Site of application and resulting potential for aquatic
exposure;
- (2) The natural geographic distribution of the microorganism;
- (3) The natural population level of the microorganism compared
with population levels likely after application;
- (4) Ability of the microbial pest control agent to survive and
replicate after application; and
- (5) The extent to which the microorganism has been manipulated
or genetically engineered.
While all of the aforementioned criteria are important, the
Agency has chosen site of application and its resulting potential
for aquatic exposure as the key criterion for establishing the ex-
tent of initial effects testing for microbial pest control agents.
The rationale for selecting this single criterion is that it direc-
ly addresses the most critical issue regarding potential hazard:
likelihood of exposure. Furthermore, criteria 2, 3, and 4 would
be illicitly considered la connection with the erittirion for site
of application. Tier II (environmental expression) testing, would
always apply in the case of genetically engineered microorganisms.
The Agency recognizes that considerable judgment will be
required ta 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 also be subjected to the ex-
panded testing. Some examples that fall into the latter category
are applications to forests, drainage ditches, riverbanks, and
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60
partially aquatic crop* such as rice. Widespread application* to
major crop* such a* cotton, soybeans, and corn could also warrant
expanded tasting. To the extant possible, the Agency will rely on
its experience with the classical chemical pesticides in distin-
guishing between terrestrial and aquatic use patterns in borde-
rline situations.
2. Tier progression, (a) Tier I. For microbial pest control
agents applied in terrestrial use patterns (where direct aquatic
exposure is not anticipated), one freshwater fish (§ 154-19) and
one freshwater aquatic invertebrate (§ 154-20) should be tested to
assess toxicity and pathogenicity. For •ierobial pest control
agents applied directly to fresh, estuarine, or marine waters, one
additional fish species and one additional invertebrate species
should be tested in Tier Z (see § 154-1, Figure 7). These tests
should be conducted as 30-day static or static renewal bioassays
using one or a combination of methods to administer the pesticide
(e.g., aqueous, dietary, or injection). 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.
No further testing would be indicated if: (1) results of
the aforementioned tests indicate no toxic or pathogenic effects,
and (2) host spectrum or beneficial insect tests indicate that the
microbial pest control agent has a narrow host spectrum such that
crossover into nontarget aquatic invertebrates is not likely. If
toxic or pathogenic effects are observed, then environmental ex-
pression testing (Tier ZZ) would follow. Zf host spectrum or
beneficial insect tests imply crossover into nontarget aquatic
invertebrates, then additional aquatic invertebrate species (those
expected to be susceptible) would have to be tested in Tier Z, or
as an alternative, Tier ZZ testing would have to be conducted. Zf
tests on additional species indicate toxic or pathogenic effects,
then testing at Tier ZZ would be indicated; if otherwise, then no
further testing would be necessary.
Genetically engineered microbial pest control agents would be
treated differently from microbial pest control agents that are
identical to naturally occurring microorganisms or that are improved
strains of naturally occurring microorganisms. Since genetically
engineered microorganisms would not have existed previously in the
environment, the more extensive data on effects to nontarget fish
and aquatic invertebrates in Tier Z (§§ 154-19 through -21) and
data on survival in the environment in Tier ZZ (ff 155-18 through
-20) are indicated in order to assess potential H&irmtmmut •!
hazards.
(b) Tier ZZ. The data for Tier ZZ are described in environ-
mental expression testing sections (§§ 155-18 through -20} of
these guidelines. Zf the environmental expression characteristics
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do nor indicate exposure of the microbial pest control agent to
nontarget fish or aquatic invertebrates, then no further testing
of these aniaals would be indicated. If Tier ZZ tests indicate
that fish and aquatic invertebrates will be exposed to- the micro-
bial pest control agent, then testing at Tier III is indicated.
(c) Tier ZZZ. Whereas Tier Z tests are designed to screen
microbial pest control agents using a •fr.l""*' hazard testing scheme,
Tier ZZZ tests are intended to sore precisely evaluate and quantify
the actual hazard associated with the microbial pest control agent.
The types of effects reported in Tier Z tests would help determine
which Tier ZZZ test(s) would be required. Zf only toxic effects
are observed in Tier Z tests, then the guidelines of H 72-1 through
-6 of Subdivision E would apply, aad further testing would proceed
as in Subdivision E. Zf pathogenic effects or both pathogenic and
toxic effects are observed In Tier Z, then tests that could be
indicated in Tier ZZZ are the following:
- (1) Additional acute or subacute test(s) of fish or aquatic
invertebrates to evaluate the spectrum of susceptible nontarget
species, or determine the susceptible route(s) of exposure, or
determine the doseresponse relationship between the pesticidal
agent and susceptible nontarget organlsm(s) (§ 154-27);
- (2) Fish embryolarvae and aquatic invertebrate life cycle
studies (§ 154-28);
- '3) Aquatic ecosystem test(s) (§ 154-29);
- (4) Test(s) to evaluate the potential for opportunistic infec-
tions ($ 154-30); and
- (5) Zn vitro studies such as tissue culture ($ 154-30).
Zf results of Tier ZZZ tests indicate no pathogenic effects,
then no further testing would be indicated. Conversely, if results
of Tier ZZZ tests, along with environmental fate data, indicate
toxic or pathogenic effects, then simulated or actual field testing
(Tier ZV) may be warranted.
(d) Tier T7. Simulated or actual field testing (§ 154-34)
provides data on the pathogenic effects of the microbial pest
control agent on fish and other aquatic animals following field
applications at actual use rates. This test would apply when
pathogenic effects are reported in Tier ZZZ testing (J$ 154-27 and
-29) at levels equal to actual or expected field'^evqpocore levels,
aad when the Agency is reasonably confident that quarantine methods
can confine the aicrobial pest control agent to the test area and
prevent contamination of adjacent areas. The specific test would
be determined on a case-by-case basis after consultation between
the Agency and the registration applicant.
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3. Major issues. This section identifies and discusses issues
regarding aquatic testing of microbial agents that require outside
input and comment. Most of the issues stem from two problems:
- (1) There are no standard widely accepted test protocols
available to evaluate the effects of microbial pest control
agents on nontarget aquatic animals; and
• (2) There are some potential hazards associated with the use of
microbial pest control agents 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.
(a) Issues associated with Tier I protocol. The desired Tier
I test protocol would simultaneously assess toxicity and pathogenicity
in aquatic animals. The maximum hazard test philosophy would be
exerted in tents of treatment level, method of pesticide administra-
tion, and age of the test animal.
A Tier I test should be conducted as a static or 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;
- (3) By injection; or
- (4) Preferably as a combination of all three routes of exposure.
If any test animals die during the test, the cause of death (e.g.,
toxicity, pathogenicity) should be determined, if possible. This
information would be used to determine what further tests, if any,
are warranted. Exposure and observation should extend for at least
30 days. Individual test animals animals should be removed
periodically throughout the test period and at test termination for
examination to assess pathogenicity.
If a sublethal 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 protocoli Committee
on Methods for Toxicity Teats with Aquatic Animals ,*4t975; Ignoffo
e- al., 1973; Var. Sssen and Anthony, 1976; Wolf, 1975; Lighcner 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.
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(1) Test organisms. The guidelines provide that the species
tested be selected from the list of species recommended by the
Committee on Methods for Toxicity Tests with Aquatic Organisms (p.
21) (1975), with the exception of goldfish. 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 for
comparative purposes;
- (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
representatives of the geographic region where the microorganism is
to be applied, and when applicable, species likely to prey upon or
scavenge the diseased target host animals should be tested.
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 a good indicator species in terms of sensitivity to
chemical toxicants;
- (2) It is partially insectivorous;
- (3) Ho other species has been shown to be preferable in terms
of sensitivity to mlcrobial pest control agents;
- (4) There is considerable background data on this species
pertaining to its mlcrobial diseases (Mann, 1978); and
- (5) Standard tissue culture procedures are available for this
species (Wolf and Quimby, 1969 and 1973).
Use of young fish (3-6 months old) is preferable since they
would be more likely to display a lethal pathogenic effect, whereas
older fish may simply carry the infection and not die.
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 phylogenetlcally similar to the
target host should be chosen. Such a test organism would be the
most likely to be susceptible to infection by the mlcrobial agent.
Therefore, when evaluating a mlcrobial agent 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.
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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 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. A potential drawback
of these species is their small size which would preclude the use
of injection as a route of exposure, although insects as snail as
adult mosquitoes can be injected with as much as one microliter of
solution. . The use of crayfish or possibly a freshwater prawn would
overcome this problem and retain the advantage of testing an animal
that would molt during the test exposure period.
(2) Method of pest control agent administration. Three
methods of pesticide administration should be considered:
- (1) Suspension in the test water (aqueous exposure);
• (2) Dietary, in the form of diseased target host animals or
incorporated into the standard feed; and
- (3) Injection.
If appropriate, and when possible, all three routes should be used
simultaneously in a single test to ensure that the susceptible
route of exposure, if any, has been tested and to challenge the non-
target test animal with the maximum possible hazard. Different
pathogens 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 microbial
pest control agents.
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 mleroblal pest
control agent. 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 (in the water) does not lower water quality to an
unacceptable level.
Dietary exposure also simulates certain natural conditions.
In fact, it is perhaps the most important means of infection for
the nonaal 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
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animals to a different life stag* of the microorganism than may be
present in the formulated product if diseased target hosts (e.g.,
insects) ara used as the feed.
An injection (e.g., intraperitoneal for fish) does not simulate
a natural route of exposure but it does insure that a known quantity
of microorganisms is brought into direct contact with the test
animal at the cellular level. Also, for many microorganisms, injec-
tion would probably be the most stringent test for infactivity.
This route may subject the test animals to some stress, but it has
been used routinely to assess microorganism effects in aquatic
animals. Protocols for the aqueous exposure, dietary, and injection
methods of administration are described by the Committee on Methods
for Toxicity Tests with Aquatic Organisms (1975)j Van Essen and
Anthony (1976); and Lightner et al. (1973), respectively. Ondeen
and Maddox (1973) also describe the injection method. Macek et al.
(1979) describe studies in which fish were simultaneously adminis-
tered chemical pesticide by aqueous and dietary exposure.
Finally, oral intubation is another possible route of exposure,
and is one that has been used to evaluate microorganism effects in
fish (Savan et al., 1979; and 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
in combination with an injection. Therefore, the oral intubation
method is not recommended.
(3) 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 life stage(s) to which nontarget
aquatic animals are most likely to be exposed. These objectives
should be achievable through the use of multiple routes of admin-
istration, provided it is known which form is most challenging and
which form is most likely to be encountered by the nontarget »«<«i«i .
The technical grade of the active ingredient (the microorganism as
it exists in the formulated product) should be used for the aqueous
exposure. The formulated product should be tested if it is to be
applied directly to water. The material to be injected should be
the form(s) or life stage(s) of the microorganism that best meets
the two aforementioned obectives (i.e., most challenging form, and
form to which nontarget animals are most likely to be exposed).
The use of diseased target hosts would beiio«t"lf»Birable for the
dietary exposure. If this is not feas£Ble*/1^!nVn"!treated feed
should be prepared, using the sane life stage(z] rhat appear in
diseased target host organisms.
(4) 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
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keeping with the maximum hazard philosophy, treatment concentrations
oust be relatively high. Consideration oust be given to the level
of exposure resulting from direct application as veil as exposure
resulting from consumption of diseased target host organisms (usually
insects), Bcposure, in terms of frequency and oumber of micro-
organisms, could be extremely high in the latter case.
Treatment concentrations for dietary exposure would be deter-
mined by the degree of infection in the diseased host. If possible,
hosts killed by the pathogen should be used to assure high numbers
of microorganisms, and the presence of any toxin that may have
been formed. Treated feed should be fortified to contain at least
Ix, and if feasible/ 2-lOx the host equivalent. (Host equivalent
is the maximum amount of active ingredient that one infected host
can contain.)
The highest feasible concentrations should be used in the
aqueous exposure and injection. At a •<«<«mimj the concentration
for aqueous exposure should equal the theoretical concentration
present in six inches of water immediately after a direct applica-
tion of the microbial past control agent to six inches of water.
A treatment concentration 100-lOOOx this level would be preferable
to impose maximum hazard and incorporate an ample margin of safety.
However, the use of such a high concentration may be limited by
its adverse effect on water quality such as oxygen depletion and
production of metabolic wastes by the microorganisms. The trea-
tment concentration used for an injection should be linked to host
equivalent. If possible, the injected material should contain a
concentration of microorganisms equal to or greater than the
adjusted host equivalent.
(Adjusted host equivalent - host x wight of test animal }
weight of host organism
In certain situations this concentration would not be feasible
and would have to be reduced. Reduced concentrations should be a
fraction of the adjusted host equivalent, in the range of 0.1-O.Sx.
Modifications to the adjusted host equivalent dosage amount should
be supported by a rationale based on sound scientific reasoning.
(5) Test duration. Exposure and observationjrast be extended
to at least 30 days (unless test animals die) to allow time for
any potential infection, microorganism replication,, or_oathogenic
or toxic effects to manifest themselves. If a snblethal infection
is observed, then the test should be extended to •vale*** the
significance of the infection. Similarly, if test animals begin
to die near the end of the 30-day period, the test should be con-
tinued to determine the fate of the remaining test population.
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67
The 30-day test duration vac selected on the basis of past
research [Savan et al. (1979); Pound (1977); Van Essen and Anthony
(1976); Lightner et al. (1973)1; and the recommendation of Suaner*
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., 20-30), it would be
logical to lengthen the period of exposure for the test animals.
Conversely, a shorter period of exposure «ay be warranted in tests
using aniaals with short life cycles (i.e., Daphnia or my* id
shrimp).
(6) Observation and examination of test aniaals. Daily
observations are required to record Mortalities and note any
behavioral, pathogenic, or toxic effects. Test organisms mist be
examined for infection or any microorganism-related effects
periodically throughout the study and at test termination. The
most difficult 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 maXe this determination include
histopathology, aerology, and nucleic acid hybridization. These
methods, and the situations in which their use may be appropriate,
were presented in the general discussion of terrestrial and aquatic
animals, part B-1.(2)(d), and in Table 1 of this Discussion.
Ondeen and Maddox (1973) used the following criteria in their
work with Nosema algerae 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 certain other microorganisms.
(b) Tier III test protocols. The embryolarvae, lifecycle,
and aquatic ecosystem tests in Tier III (§§ 154-28 through -29)
would follow the same general protocols that are referenced for
these types of tests in Subdivision E of the guidelines (§§ 77-4
through -7). However, generally accepted standard protocols for
conducting these studies with microbial pest-control agents have
not been developed. In fact, few, if any, such tests have ever
been conducted with microbial pest control agents* 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 needed
in this area before the Agency can maXe specific recommendations
concerning protocols and tier progression.
(c) The role of in vitro and Tier IV testing. The Agency
recognizes that there are in vitro tests available to assess the
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inactivity ot 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 infactivity in certain situations. However,
the relationship between effects demonstrated by ia vitro tests
and effects likely to occur under ^n vivo situations is uncertain.
For example, Ignoffo (1975) states that "Tissue, completely non-
susceptible in the intact organism, may support viral Multiplica-
tion 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 Z. 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 infactivity in a broad spectrum of species; and
- (2) As a test to support or check the results of _in vivo tests.
Therefore, a provision for tissue culture studies is included in
Tier ZZZ of the testing scheme.
(d) Tier ZV Testing. The Agency recognizes the possible
danger in using simulated or actual field tests (Tier ZV) as the
final test of the safety of a microbial pest control agent. Zf 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 previously conducted laboratory tests. This fact would
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 cause widespread environmental
damage, unless adequate quarantine measures could be taken. There-
fore, before any Tier IV field test is to be undertaken, the appli-
cant should discuss its plans with the Agency concerning potential
hazards. If the Agency determines that a Tier TVrfield test would
pose an unacceptable risk, then the microbial pest control agent
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 microbial pest control agents that demonstrate no
hazard in Tier Z 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
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69
-testing, and the Agency would strongly encourage »uch testing. This
would provide the opportunity to evaluate pesticidal effects (both
direct and indirect) on a much broader spectrum of nontarget species
than is.possible in Tier Z testing.
(«) Assessment of other potential hazards; opportunistic
infections and latent viruses. Opportunistic infections in nontar-
get aquatic animals are recognized by the Agency to be a potential
hazard. X similar concern is noted for the formation of latent
viruses. Research indicates that aquatic animals may be rendered
significantly more susceptible to mierobial infection, (e.g., by
viruses and bacteria) when stressed by such factors as Aroclor
1254 (Couch and Courtney, 1977), copper (Hetrie* et al., 1979),
temperature, salinity, pesticides, and other pollutants, (Snieszko,
1974, and Schwartz, 1974). This increased susceptibility raises
several important questions:
- (1) What is the likelihood of an opportunistic infection (from
a mierobial past control agent) occurring in an abnormal host
such as a nontarget aquatic animal?
- (2) Will the proposed Tier I test adequately screen mierobial
pest control agents for potential opportunistic effects? Or
could a mierobial pest control agent be non-infective 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?
There is far too little background information and research on
mierobial pest control agents 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 both sublethal infections and latent viruses produced in Tier
I tests can be detected if the proper methods of detection are
employed. However, the potential for an apparently non-infective
agent (in Tier I testing) to infect stressed animals is unknown.
And, 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 microorganism/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 mat 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.
(f) Oneoqenic effects. The Agency recognizes the potential
for oncogenic effects that are associated with viruses. The
probability of oncogenicity in nontarget aquatic animals, as a
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70
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. 1975k Methods for Acute Toxicity Tests with Pish,
Macroinvertebrates, and Amphibians. U.S. Environmental Protection
Agency. Ecol. Res. Series. EPA 660/3-75-009. 66 p.
(2) Couch, J.A., M.D. Summers, and L. Courtney. 1975.
Environmental Significance of Baculovirus infections in estuarine
and marine shrimp. Annals N.Y. Acad. Sci. 196:528-536.
(3) Couch, J.A., and L. Courtney. 1977. Interaction of
chemical pollutants and virus in a crustacean: A novel bioassay
system. Annals N.Y. Acad. Sci. 198:4977-504.
(4) H«trick, P.M., M.D. Knittel and S.I,. Pryer. 1979.
Increased susceptibility of rainbow trout to infectious hematopoietic
necrosis virus after exposure to copper. Appl. and Envir. Kicrob.
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. Soc. Microbiology.
Washington D.C.
(6) Lightner, O.V., R.R. Proctor, A. 1C. Sparks, J.R. Adams,
and A^M. Heimpel. 1973. Testing Penaeid shrimp for susceptibility
to an insect Nuclear Polyhedrosis virus. Environ. Ent. 2(4):611-
613.
(7) Macek, K.J., Macefc, J.K., S.R. Petrocelli, and B.H.
Sleight III. 1979. Considerations in assessing the potential for,
and significance of biomagnification of chemical residues in aquatic
food chains. ASTM, STP. Pp. 251-268, American Society for Testing
and Materials, Philadelphia.
(8) Mann, J.A. 1978. Diseases and parasit** >bf fishes: An
annotated bibliography of books and symposia, 1904-1977. Pish
Disease Leaflet 53. OSDI. Pish and Wildlife Service, Washington,
O.C.
(9) Narayan, K., R. Govinarajan, S. Jayaraj, S.P. Raj, and
M.N.Kutty. 1977. Non-susceptibility of comoon carp, Cyrinus earpio
L. to nuclear polyhedrosis virus, Baculovirus amsacta of groundnut
red hairy caterpillar. Madras Agric. J. 642(6)i411-412.
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71
(10) Pound, J.G. 1977. Safety and potential hazards of the
entoraopathogen Mattesia troqodermae to nontarget species. Ph.D.
dissertation. University of Wisconsin.
(11) Savan, M., J. Budd, P.M. Reno, and S. Darley. 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.P. 1974. The effects of environmental
stress on outbreaks of infectious diseases of fishes. J. Fish.
Biol. 6:197-208.
(14) SuoBers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
1975. Baculoviruses for Insect Pest Control: Safety Considerations.
Selected papers from EPA-0SDA Working Symposium. ••American Society
for Kicrobiology. Washington, D.C.
(15) Surtees, G. 1971. Epidemiology of microbial control on
insect pest populations. Intern. J. Bnv. Studies 2:195-201.
(16) Ondeen, A.H., and J.V. Maddox. 1973. The infection of
non-mosquito hosts by injection with spores of the microsporidan
flosema algerae. J. Invert. Path. 22:258-265.
(17) Van Essen, F.W., and D.W. Anthony. Susceptibility of
nontarget organisms to Nosema algerae (Hicrosporida: Nosematidae),
a parasite of mosquitoes. J. Invert. Path. 28:77-85.
(18) Wolf, K. 1975. Evaluation of the exposure of fish and
wildlife to nuclear polyhedrosis and granulosis viruses. In:
Baculoviruses for Insect Pest Control: Safety Considerations, M.
Summers et al. (eds.) Am. Soc. Mlcrobiol.
(19) Wolf, K., and M.C. Quimby. 1969. Fish Cell and Tissue
Culture. Pp. 253-305 in Fish Physiology. Vol. 3. W.S. Moor and
D.J.Randall, eds. Academic Press, Mew York.
(20) Wolf, K., and M.C. Quimby. .1973. Towards a practical
fail-safe system of managing poikiiothermic vertebrate cell line
culture. In Vitro 8:316-321.
B-4. Nontarget Plant Testing.
1. Approach. The plant testing scheme proposed herein is based on
the tier testing scheme found in Subdivision J of the registration
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72
guidelines (Hazard Evaluation: Nontarget Plants). Those guidelines
and their accompanying preamble should be perused with respect to
the plant tier testing scheme, the tests, and the dose.
The Agency has determined that phytotoxicity data for conven-
tional chemical and biorational biochemical pesticides will be
requested only on a case-by-case basis. He have based this on the
premises that these substances readily degrade, their long-tem
effects are minimal, and that most off-target effects are due to
environmental and/or application equipment factors. On the other
hand, the Agency has determined that phytotoxicity studies should
be submitted for all microbial ag*nt pesticides. The pesticide
science for these agents is relatively new and their adverse effects
can be far-reaching. These effects may occur because the microbe
is capable of extensive regeneration in a favorable environment.
If it is transported to other off-target areas and the selectivity
is not known, considerable damage to desirable plants can occur.
2. Discussion and major issues. Plants and animals of an undis-
turbed natural system exist within a narrow balance. This balance
is maintained by competition for space and by the biological con-
trol of those organisms that might become pests to the system.
During a disturbance of the- balance, a biotic entity for which
natural enemies in that area do not exist could be introduced and
become dominant. Such is the case with a number of plants that are
now destructive weeds and such could be the case for introduced
organisms that man may use to attack these weeds. However, suffi-
cient surveillance and careful examination of such introduction
can prevent the adverse effects to the extent that introduced
weeds are causing.
Of great economic and natural threat to a majority of the
aquatic areas of Florida and one which is spreading to aquatic
areas from California to Virginia is the water hyacinth, Sichomia
crassipes. A biological control agent has been found for this
menace in Cerospora rodmanii, a fungus. It is highly specific,
easily disseminated, and has not been found to affect man, wild-
life, fish or domestic animals (Freeman, 1977). It is so specific
that, in tests with over 80 higher terrestrial and aquatic plants,
there was no infection of healthy plants (Conway and Freeman,
1977). Unhealthy plants may, however, be detrimentally affected.
A request for an experimental use permit has been granted by the
ZPA for testing in Florida and Louisiana.
Algal blooms have destroyed once clear lakes and .ponds through-
out the united states because of Introduced rmrrqjmj| nation. The
various algal blooms are in many instances being partially con-
trolled by agents such as viruses that are cultured and employed
as microbial pest control agents. A number of the viruses have
been identified along with their respective host(s) (Brown, 1972).
One of the better known and first to be identified is LPP-1 which
attacks species of Lyngbya, Plectonema, and Phormidium.
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The primary concerns with respect to the use of living
organisms on or near desirable or nontarget plants in the control
of pest plants are the selectivity of the organisms, the purity
of the organism or strain produced (quality control), and the
persistence or lack thereof of the organism. Both Freeman (1977)
and Brown (1972) stated several criteria that must be satisfied in
varying degrees in order for the pathogen to be a desirable candi-
date as a biological control agent for pestiferous plant species*
These criteria include:
- (1) Selectivity for the specific pest organism;
- (2) Absence of adverse effect on man;
- (3) Absence of adverse effect on domestic animals, fish, wild-
life, and desirable insects;
- (4) Absence of adverse effect on nontarget or desirable plants;
- (6) Absence of any detrimental effects on water quality;
- (7) Lack of accumulation in non-target organisms;
- (8) Base of production, dissemination, and self-maintaining
when established;
- (9) Effectiveness under the environmental conditions of the
intended use locations; and
- (10) Simplicity of assay for its presence in small amounts,
both quantitatively and qualitatively.
With respect to selectivity, the most important consideration
is that a number of strains of the pesticide organism may exist,
each being selective for an individual or group of organisms.
Therefore, when an organism is mass-produced and used in areas
other than its original habitat, the organism must be tested for
its selectivity not only to the specific pest plant but also to
closely related (same genus or family) nontarget plants.
Testing procedures similar to those found in the Subdivision J
guidelines, i.e., the testing sections for both the target areas
(§ 121-1) and nontarget areas (§§ 122-1 through 125-4), would be
used. (See $ 154-1, Figure 8.) Plant species and varieties similar
to the pest and to the desirable target area plants ^ould be tasted
in addition to a basic set of plants for possible susceptibility.
During mass production of live organisms to be used for pest
control, other strains may develop or chemical by-products may be
introduced that are injurious to plants in general. Quality control
testing is necessary to determine any detrimental effects of the
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end-products of the production process. This tasting may be done
by chemical analyses (primarily for toxic chemical by-products) and
biological analyses for purity of the end-product. The biological
analysis would have to be tailored to each organism to be tested
but would follow the applicable guideline* as found in Subdivision
J.
Zt may be desirable for the pesticide organism to have some
persistence characteristics. Such characteristics would include
ability to survive introduction and to grow and reproduce in
sufficient numbers that the organism will be efficacious. Where
persistence occurs due to sporulation, detrimental effects of the
viable spores and their products would need to be determined.
Testing for the persistence of the pesticide organism which might
exist in the presence of nontarget plants and for any detrimental
effects due to this quality would need to be conducted in a manner
similar to the selectivity testing noted above.
In the testing regime, one of the considerations with respect
to uses for pest tn1"»*T control is whether the material is to be
used within its area of natural occurrence. Where a microbial pest
control agent is proposed for use in an area where it does not
naturally occur/ tests would be performed to determine if the
microbial pest control agent is phytotoxic and, if so, the extent
of the effect.
The ultimate test in all of these schemes would be the evalua-
tion of these microbial control agents for nontarget plant resis-
tance and susceptibility under field conditions. Conway and Free-
man (1977) have stated that because many plants do not show defined
responses to a pathogen, criteria are needed to evaluate the degree
of damage necessary to classify a fungus (or other microorganism)
used for biological control as a threat to other crops and nontarget
plants. The data supplied as proposed by these guidelines should
provide the necessary information to meet this criterion for the
evaluation process.
Literature Cited
(1) Brown, R.M., Jr. 1972. Algal viruses. Adv. Virus Res.
17:243277.
(2) Conway, K.X., and T.E. Preedman. 1977. Host specificity
of Cerospora ro**«Tii, potential biological control of water hyacinth.
Plant Pis. Rep. 61(4)s262-266.
(3) Freedman, T.E. 1977. Biological control of aquatic
weeds with plant pathogens. Aquatic Botany 3(2):175-184.
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B-5. Nontarget Insects.
1. Terrestrial insects, (a) Approach. Assessment of potential
nontarget insect hazard from uses of microbial pest control agents
is made difficult by a number of factors:
- (1) Most microbial pest control agents 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) There are very few nontarget insects whose importance to
man can be measured economically. There are many species whose
Importance (e.g., in a food chain, or in regulation of population
levels of a crop pest) may be difficult to justify the expendi-
tures necessary for testing and evaluation.
- (3) Unlike chemical pesticides, most microbials will work
through pathogenicity rather than toxicity. The simple, short,
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 microbial agent for infectivity and for its ability
to reproduce or develop in the test insect.
- (4) Hazard evaluation for a microbial pest control agent will
involve determination of the host range as an important factor.
A problem here is that extrapolation, even across species lines,
is often not dependable. For this reason, the Agency will
provide for testing with representatives 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.
The Agency is aware that this first tier of testing may, in
some 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 baseline level.
In view of the factors cited above, the tier-tasting scheme
for microbial past control agents is based on a fairly extensive
first tier. The purpose of the Tier I testing is to assess toxicity
and pathogenicity of the microbial agent 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
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such factors as th« likelihood of exposure to the microbial agent,
phylogenetic proximity of the test species to target pest species,
and similar relationships. A rationale for selection should be
developed by the registrant.
Testing beyond Tier I, trill vary depending on whether or not the
microb'ial pest control agent is * genetically-engineered microorgan-
ism, while further testing for non-genetically-engineered microorgan-
isms will depend on results of Tier I testing, genetically-engineered
microorganisms will automatically go to Tier II testing (environmental
expression). Beyond this point, the testing for the two types of
microbial pest control agents is substantially similar. The testing
scheme is discussed in detail detail in the following sections on
tier progression.
(b) Tier Progression. (1) Tier I. Under these guidelines,
toxicity/ pathogenicity tests on the honey bee and on three species
of insect predators and/or parasites are indicated for all microbial
pest control agents (see § 154-1, Figures 9 and 10). Selection of
predator and parasite species for testing is made by the registra-
tion applicant. Species selected should be representative of groups
which will be exposed under the conditions of proposed use, and which
have some important relationship 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 in-
secrs. As noted above, the representative test species selected, in
addition to the honey bee, should be of some importance in the agro-
ecosystem to be exposed to the microbial control agent* Data derived
from Tier I testing will be used in conjunction with available infor-
mation on use pattern/ host rang* (specificity), fate, and other
similar factors, to assess potential for adverse effects. If data
indicates 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 ex-
pression} would follow.
For all genetically-engineered microorganisms, testing includes
appropriate Tier I tests and Tier II (environmental expression)
testing.
(2) Tier II. The data for Tier II are described in environ-
mental expression testing (§ § 155-15 through -23) of these guidelines.
For nongene tic ally-engineered microorganisms, ^.safpreesion character-
istics preclude exposure, no further testing wgold be.Indicated. If
data indicate that nontarget insects will be exposed to the microbial
pest control agent, then the registration applicant should consult
with the Agency regarding possible Tier III testing.
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For «11 genetically-engineered microorganism*, available infor-
mation on us« pattern, host range, and other similar factors should
be used in conjunction with the Tier Z and Tier II data to access the
oicrobial agent's potential for adverse effects on nontarget insects.
If no adverse potential is indicated at this point, no further test-
ing would be indicated. If data indicate that the potential for haz-
ard exists, data on expression characteristics should be closely
examined to determine exposure potential. If expression characteristics
do not indicate exposure, no further testing would be indicated. De-
termination that certain susceptible nontarget insects would be exposed
should usually lead to consultation with the Agency regarding further
testing or registrability of the product.
(3) Tier III. For all microbial pest control agents, Tier III con-
sists 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.
2. Aquatic Insects, (a) Approach. Tier I testing, as outlined
in the "Aquatic Animal Tier Testing Scheme for Microbial Pest Control
Agents" (§ 154-1) will include toxicity/pathogenicity testing with
Daphnia, or a species of aquatic insect, or both, depending on use
pattern. Detection of pathogenlcity/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.
VIII. ENVIRONMENTAL FATS AND EXPRESSION
A. Biochemical Pest Control Agents; Environmental Fate Testing.
1. Scope and approach. The term environmental fate pertains to
biochemical pest control agents (whereas the term environmental
expression pertains to microbial pest control agents). The purpose
of environmental fate testing is to generate the data necessary to
estimate the concentration of a biochemical pesticide and its
degradates occurring in or on various smflia (i»e«/ qo4j>«'»**ter,
air) at intervals after pesticide application. Generally these
data would be submitted if adverse effects are observed in Tier I
environmental effects tests or if the biochemical is applied directly
to water. Figure 4 outlines tne KnviroosMntal Fate testing
philosophy.
Certain studies in Subdivision M refer to Subdivision N tests
that specify identification of any degradation products comprising
more than ten percent of the initial concentration, or in some
cases levels greater than 0.01 ppm (section series 161 and 162).
These studies are hydrolysis, photodegradation (soil, water, and
vapor phase), aerobic soil metabolism, and aquatic metabolism
studies. The Agency recognizes that application rates of many
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biochemical pesticides nay be so low chat identification of degra-
dation produces, or even calculation of percent parent material
renaming, may be difficult at best. Therefore, the guidelines
provide that biological monitoring techniques may be substituted
for quantitative chemical analysis by instrumental methods.
Specifically, this approach could be used:
- ( 1 ) To demonstrate the decline in concentration of the initially
applied parent pesticide material, target organisms could be
used in a bioligical monitoring test to demonstrate pesticide
decline in a manner similar to efficacy testing;
- (2) To demonstrate the safety of a degradation product (mixture
material and/or degradates and/or metabolites ) , biomonitoring
tests aimed at making this determination should employ a
test design similar to that required for Tier Z toxicology
testing (i.e., rat acute oral
This is a novel approach to the problem and in many situations
may be less expensive than instrumental analysis. However, standard
accepted protocols for this testing approach are few, particularly
regarding biomonitoring tests to demonstrate safety. Further
discussion of this approach is presented in the discussion section
that follows.
2. Discussion. The Tier II environmental fate testing scheme
consists of twelve separate tests. Seven of these tests are
identical to those described for conventional chemical pesticides
in Subdivision N or Subdivision 0* The remaining five tests are
new. These tests, (volatility of dispensed product, dispenser-water
leaching, DV (ultraviolet) absorption spectra, biomonitoring for
degradation products, and biomonitoring for disappearance of bio-
chemicals) address some of the unique properties of biochemical
pesticides.
The following discussion pertains only to the five test methods
unique to this subdivision. The discussion includes justification
for each of the selected test methods, use of the data, and specific
problems the Agency recognizes in conducting these tests.
(a) Volatility of Dispensed Product (i 155-4). „( 1j^. -Choice
of Method. Volatility data are difficult to interpret due to
inability to easily control such variables as temperature, surface
area, and flow rate. An ideal assessment of volatility
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Figure 4—ENVIROWtENTAL FATE TESTING PHILOSOPHY
Potential adverse effects are'seen in Tier I testing. (If not/ no
testing is indicated in Tier II unless product has aquatic use pattern or
biocheaical is not applied in a controlled release device, in which case
tester should proceed directly to Persistence Testing.)
V
TRANSPORT TESTING:
Volatility (§ 155-4)
Dispenser water leaching (§ 155-5)
Vapor pressure (| 151-17)
Hater solubility ($ 151-17)
V
Estimated Environmental Concentration (EEC) determination from
mass-balance analysis.
I
V
EEC indicates potential hazard. (If not, no further testing is indicated.)
V
PERSISTENCE TESTING:
Adsorption/desorption (§ 155-6)
Octanol/water partition coefficient (§ 155-7)
Hydrolysis (§ 155-9)
Aerobic soil metabolism (§ 155-10)
Aerobic aquatic metabolism ($ 155-11)
Soil photolysis (§ 155-12)
Aquatic photolysis ($ 155-13)
V
potential for adverse effects to nontarget organisms,
(If not, no further testing is indicated.)
V
Additional nontarget organism testing is indicated (Tier III)
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- (1) Micrometeorological factors (temperature, wind
rainfall);
- (2) Degradation of biochemicals inside dispensers in uncon-
trolled environments, if applicable;
- (3) Changing rates of biochemical diffusion to dispenser
surfaces, if applicable;
- (4) Vapor phase degradation; and
- (5) Adsorption to plant leaves.
The proposed method combines field and laboratory testing,
allowing the assessment of factors (1), (2), and (3) above. Vapor
phase degradation (4) can be measured and accounted for as described
in the discussion on UV-visible absorption measurements. [See
paragraph (c), below.] Unfortunately, the present state of the art
on these compounds (e.g., semiochemicals) prohibits consideration
of factor (5).
(2) Dse of the data. Volatility data will be used in two
ways. The data will be evaluated along with dispenser water leaching
data, if applicable, to predict the extent of loss of biochemicals
to soil or water. For example, if the volatility of a formulated
biochemical is very high and the water leaching very low, it would
be unlikely that a large percent of the biochemical would reach the
soil. The data will also be used to estimate vapor phase concentra-
tions near sites of application in cases where there is concern
regarding beneficial insects and/or reentry hazards. Volatility is
a complex issue, and the Agency invites comments on the method pre-
sented in § 155-4.
(b) Dispenser-water leaching ($ 155-5). (1) Choice of
method. Standard methodology is not available which stimulates
rainfall leaching biochemicals from dispensing devices. However,
the method provided is one that has met with sane agreement among
Agency and outside experts. Although it is a novel approach, the
chemistry involved is simple and classic. The method involves an
eight-hour exposure of the formulated pesticide to water, followed
by quantitative analysis.
Eight hours was chosen as the leaching extraction time for two
reasons. First, an eight-hour exposure to water seems to be a
reasonable simulation of heavy, prolonged rainfall* Second, a few
hours is generally sufficient time for liquid-liquid extractions.
For example, DSZPA (1979) recently recommended a one-hour extraction
time for the octanol/water partition coefficient. If the dispenser
is constructed such that it is at least partially permeable to water,.
then liquid extraction will occur/ since the biochemically active
ingredients are usually dissolved in organic inert a.
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(2) Use of th« data. Dispenser-water leaching data will be
used with volatility data to assess the potential for leaching of
active ingredients to water or soil. Zf the potential for leaching
is great (i.e., the estimated environmental concentration is greater
than 1/5 LCgg or K$Q of the exposed non-target species), further
testing may be indicated.
(c) Ultraviolet-Visible Absorption Spectra (} 155-8). (1)
Choice of methods. The methods required are very similar to those
previously described by EPA (1979) in testing guidelines for new
chemical substances under the Toxic Substances Control Act (44 FR
16240). The vapor phase absorption spectrun is preferred, but the
liquid phase spectrum (in inert solvents) is acceptable. The
methods are simple and inexpensive, and they yield useful data.
(2) Use of the data. OV-visible absorption spectra data will
be used in the prediction of vapor phase half-lives when there is a
potential for harm to beneficial insects. The three predominant
means of atmospheric transformation are photolysis, reactions with
hydroxyl radical, and reactions with ozone. Photolytic half-lives
can be estimated in the following manner. The OV-visible absorbances
are converted to absorption cross sections. These data are then
used in combination with the solar flux and an estimate of the
quantum yield to calculate the photolytic half-life. Transforma-
tion products can often be predicted, if necessary, by the scheme
developed by Hendry and Kenley (1979). Rates of reaction and
formation of transformation products resulting from reaction with
hydroxyl radicals and ozone may also be predicted using the approach
described by Hendry and Kenley (1979).
(d) Biomonitoring for disappearance of biochemicals. (1)
Choice of Methods. Generally techniques very similar to those used
in efficacy assessments (see section series 156) can be used to
follow disappearance of biochemicals. Test methods will vary with
the sites and the pests. Some relevant test methods are contained
in volumes 1, 2, 3, 4, 8, and 10 of an American Institute of
Biological Science (AIBS) report to EPA (1977, 1978). Pheromone
activity as has been determined with the electroantennogram method
(SAG) (Am, 1975; Schneider, 1969) may not be appropriate for
monitoring activities of mixtures, since only isolated peaks are
examined with GC-KAG. Bioassay methods for detecting plant growth
regulators in soil and water are contained in a report by Mitchell
and Livingston (1968).
(2) Dse of the data. The data can be OMd for determining
half-lives of biochemicals in lieu of instrumental determinations.
(e) Biomonitoriag for degradation products. (1) Choice of
methods. Determination of all degradation products exceeding 10
percent of the initial pesticide concentration (as in portions of
the Tier II tests that refer to tests in Subdivision N of the
7uidelir.es) can be time-consuming and costly, and may generate data
that are difficult to interpret toxicologically. Thus the guidelines
allow registrants to test concentrated mixtures of transformation
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products in the Tier I human health effects screen (§§ 152-10
through -18). If significant adverse effects are detected, further
identification of transformation products may be warranted. These
alternative methods apply to hydrolysis, aquatic photodegradation,
and aquatic metabolism studies.
An established method for concentrating a mixture of degradation
products in a toxicologically and chemically significant manner is
not available. The guidelines method solicits performance of
environmental fate tests at much higher concentrations than would
normally be used in such studies. Zt is necessary to raise the
concentration of these studies because maximum concentrations
required in effects tests are generally higher than the maximum
concentrations required in environmental fate studies.
(2) Dse of the data. The data from either test chosen to be
performed will be used to determine the toxicological significance
of degradation products identified in environmental fate testing.
The Agency welcomes comments on the relative cost effectiveness of
the biomonitoring and bioassays as alternative approaches (relative
to instrumental identification of reaction products) asz well as
comments on their scientific merit.
(3) Tier progression. A biochemical pesticide will reach
Tier II, environmental fate testing, because either.
- (1) The biochemical will be introduced directly into an aquatic
environment (i.e., direct application to water), or
- (2) Adverse effects on non-target organisms are observed in
Tier I.
Literature Cited.
(1) Abdel-Rahman, M., B.A. Schneider, and J.R. Frank. 1977.
Plant Growth Regulator Handbook of the Plant Growth Regulator
Working Group. Great Western Sugar Co., Agricultural Research
Center, Sugar Mill Road, Longmont, Colorado 80511.
(2) Anonymous. 1979. Steroid plant growth promoter identified.
Chem« Engin. News Pg. 20. Nov. 5, 1979.
(3) American Institute of Biological Sciences (AIBS). Analysis
of Specialized Pesticide Problems Invertebrate Control Agents -
Efficacy Test Methods. Volumes 1, 2, 3, 4, 8,~'«W">1(Ji EPA contracts
540/10-77-001, 540/10-77-007, 540/10-77-OOJ, 540/^7t-006, and
540/70-78-002, respectively, 1977 and 1978.
(4) Am, Hw, E. Stadler, and S. Raucher. 1975. The electro-
antennographic detector - A selective and sensitive tool in the gas
chromatographic analysis of insect pheromones. Zeitsehrift
Naturforsehung 30:772-725.
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83
(S) Bierl-Leonhardt, B.A. , E.D. DeVilbiss, and J.R. Plimmer.
1979. Rate of release of disparlure from laminated plastic dispen-
sers. J. Econ. Entomol. 72(3) : 319-321 .
(6) Hendry, D. and R.A. Kenley. 1979. Atmospheric Reaction
Products of Organic Compounds. EPA contract 560/12-79-001. May
1979.
(7) Inscoe, M. 1977. Chemical communication in insect*. £._
Wash. Acad. of Sci. 67(1): 16-33.
(8) Lawrence, J.F. and D. Turton. 1978. High performance
liquid chromatographic data for 166 pesticides. J. Chromatography
159:207-226.
(9) Mackay, D. 1979. Transfer rates of gaseous pollutants
between lakes and the atmosphere. 178th Meeting of the Amer. Chem.
Soc. Sept. 9-14, 1979. Washington, D.C.
(10) Mackay, D., and T. K. Yuen, (in press-).. Transfer rates
of gaseous pollutants between the atmosphere and natural waters.
In Proceedings of ACS Symposium on Atmospheric Input of Pollutants
to Natural Waters. S.J. Eisenrich, ed. A&er. Chem. Soc., Washington
D.C.
(11) Macfcay, D., and T.K. Yuen. 1980. Volatilization rates
of organic contaminants from rivers. In Proceedings of 14th Canadian
Symposium, 1979. Hater Pollution Research, Canada.
(12) Mitchell, J.W., and G.A. Livingston. 1968. Methods of
Studying Plant Hormones and Growth-Regulating Substances. Agriculture
Handbook Ho. 33. Agricultural Research Service. USD A. , Washington,
O.C.
Plinnner, J.R. , J.H. Caro, and H.P. Freeman. 1978.
Distribution and dissipation of aerially-applied disparlure under a
woodland canopy. J. Eeon. Entomol. 71 (2) : 155-157.
(14) Schooley, David. 1979. Personal communication. Zoecon
Co., Palo Alto, Calif.
(15) Thimann, X.V. 1972. The Natural Plant Borsones. Pg.
359 in Plant Physiology: A Treatise. F.C. Steward, ed. VI B, S.
(16) OSEPA (Environmental Protection Agency). 1979. Discus-
sion of Premanufacture Testing Policy and Technical Issues? Request
for Comment. Fed. Reg. 44 ( 53) : 16240-16292. March 16, 1979.
B. Xierobial Pest Control Agents; Environmental Expression Testing.
1. Introduction. The guidelines employ the term "environmental
expression" to be used with microbial pest control agents in order
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to distinguish th«m from chemical pesticides and the term "environ-
mental fate." Environmental expression is defined as the ability
of the physiologically active component of the microbial pest
control-agent to propagate and become established in a new niche
or host after it has been introduced (applied). The term "fate,"
on the other hand, has been associated with chemical pesticides
and is defined in the riFRA guidelines as the transport and trans-
formation of a chemical by natural means after it is released to
the environment from a point source, disposal site, and/or disper-
sive use. This definition does not apply to microbial pest control
agents and, therefore, the guidelines set forth the above distinc-
tion and use .of the term "environmental expression."
The tier testing scheme in section series 154 and 155 of Sub-
division M is designed to present the ««*^»mjri hazard to nontarget
organisms in the initial (Tier I) testing of microbial pest control
agents. When the Tier I navlnmin hazard tests yield negative results,
no further testing is indicated and thus testing is kept to a
minimum. Positive results (toxic or pathogenic effects) observed in
Tier I will mandate further testing in Tier IX, environmental
expression testing.
Tier II consists of screening tests that will eliminate the
need for Tier III testing whenever the results show that the agent
will not become permanently established once applied or inadvertantly
inserted into a new niche or host, or cannot survive except under
special conditions (e.g., specific host, obligatory heterotroph).
Data development in Tier III testing requires methods that
approach the state of the art in microbial ecology. Without Tier
II testing, positive results in Tier I would require that the
registrant proceed to the more sophisticated Tier III testing,
which would probably be very costly. Thus Tier II tests can
eliminate the need for intensive Tier XII tests for many agents.
In Tier II, the agent is tested for ability to persist in a
terrestrial, freshwater, marine, or estuarine environment so that
potential exposure of nontarget organisms can be determined. For
example, an agent could be saprophytic (i.e., dependent on decaying
matter for nutrition in its normal environment), but could proliferate
independently and unchecked in a new niche. If^tjcan be determined
that the application of the microbial pest control 'agent will not
give it an opportunity to proliferate in a new niche, then no
further testing is needed. The reason it cannot survive and grow
may be the lack of an obligatory nutrient, or an absence of suitable
hosts other than the target organism. If, however, there is a
possibility of insertion of the microbial pest control agent into a
new niche where no natural antagonists of the agent are known, then
Tier -III- testing must be done to determine-the possible toxic or
pathogenic effects of such insertion.
Actual field testing (Tier IV) may be necessary to decide
whether the agent can proliferate in a given environment, but
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because the agent is capable of permanent persistence (a property
not demon*trated by chemical agents), such testing should only be
undertaken after the registrant and the Agency have carefully
considered the possible consequences.
The registrant nay be able to identify chemical or physical
markers with which the presence of the agent can be detected. Such
markers could be either in the host or the environment. For example,
standard laboratory procedures employ bioassay for quantitative
assay of viruses, but these depend on the infective potency of the
agent. However, in measuring potency of an industrial product or
screening field samples, bioassay nay be useless because the virus
fora being studied is one which is not infectious to tissue culture
cell lines. There is great need for rapid and unequivocal iden-
tification of such virus forms in field samples or in any scheme
designed to monitor the environment after application of a pesti-
cidal virus. Reserach in the area may lead to development and
distribution of type-specific antisera to pest management workers
who can make checks on virus identity with unsophisticated equip-
ment.
2. General information. This section outlines the scope and
approach for Tier ZZ microbial testing.
(a) Tier ZZ testing. A microbial pesticide will reach Tier
II:
- (1) Whenever Tier I testing gives positive results (toxic or
pathogenic effects) in maximum hazard testing; or
• (2) Whenever the agent is a genetically engineered organism.
(b) Approach. Use pattern (terrestrial, freshwater, and/or
estuarine/marine) in conjunction with positive Tier I test results
will determine which one of three environmental expression tests
should be conducted under Tier II testing. The tests include a
greenhouse test for determining expression in a terrestrial
environment and two aquaria tests for determining expression in
freshwater and in estuarine/marine environments.
IX. PRODUCT PERFORMANCE DATA REQUIREMENTS
Product performance (efficacy) data requirements hare been
generally waived for most products. It should be noted, however,
that the Administrator may require, on a case-by-case basis/
efficacy data on any specific product whenever he deems that such
ISita are necessary to make Jpr'opTr evaluations zbr oecisions" "as"To"
acceptability to register or to maintain or cancel registrations.
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Certain efficacy-related data on biorational pest control
agents have been solicited in the** guidelines. These data would
ordinarily be subject to the waiver policy: the available informa-
tion 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 considered important in the evaluation of nontarget
organism safety data concerning biorational pesticides.
X. EXPERIMENTAL USE PERMITS
Data to support applications for experimental use permits for
biorational pesticides, as described in section series 157, generally
include those data that would ordinarily be developed first in
preparation for product development and registration. 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 prepar-
ation for registration. Unless these test results indicate toxic,
pathogenic, or other harmful properties, no data on residues or
environmental fate would be necessary. Efficacy data follow the
pattern already proposed in the Subdivision I guidelines, which
waives the requirements for most products not dealing with public
health areas, but § 157-4(g) also proposes the submittal of data on
host spectrum, and time and minimum effective dosage required to
achieve the product performance standard.
XI. PROMOTING BIORATIONAL PESTICIDES BY MODIFYING LABEL CLAIM
REQUIREMENTS
Consistent with the Agency's mandate'to "protect health and
the environment," the EPA is currently pursuing avenues to promote
the development and use of biological control agents. With publi-
cation of "Regulation of 'Biorational1 Pesticides; Policy Statement
and Notice of Available Background Document" (44 F* 23994, May 14,
1979), the EPA recognized "that biorational pesticides are inher-
ently different from conventional pesticides" and that "the funda-
mentally different modes of action of biorationals and the conse-
quent lower risks of adverse effects from "their use* must be taken
into account. Embracing this'policy, with the development of
Subdivision M guidelines, the Agency has endeavored to reduce the
burden of extensive data development by the introduction of the
tier testing concept. This departure from standard procedures is
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intended to function as * catalyst for development of additional
innovative control agents. Those parties interested in the develop-
ment of the Agency's rationale supporting the direction of its
proposed policies and guidelines may refer to the above noted Federal
Register Notice.
In addition to those concepts expressed within the Biorational
Policy Statement, Sections 20(a) and 28 of the amended FIFRA mandate
the Agency to a role supportive of biorational pesticide use.
Sections 20(a) and 28 of the Act express Congress' intent for the
promotion of Integrated Pest Management (IPM) and "the safe use
and effectiveness of chemical/ biological and alternative methods
to combat and control pests...." In response to Congressional
wishes, the Agency has moved forward with the funding of applicable
research, the reduction of data requirements and the expeditious
processing of registration applications for biorational pesticides.
The Agency recognizes, however, that thus far it has only
partially tapped its arsenal of regulatory alternatives available
for the promotion of those methods compatible with the desires of
Congress. Public awareness of the unique qualities inherent in
biorational pesticides is an'integral element to the successful
promotion of these agents for practical use. One of the more
obvious vehicles available for reaching the public is pesticide
labeling. Declaration of certain unique characteristics of these
agents, those responsible for their special recognition, is cur-
rently prohibited by 40 CTR 162.10(a)(5)(ix). This section pro-
hibits "claims as to the safety of [a] pesticide or its ingredients,
including statements such as 'safe,' 'nonpoisonous,' 'noninjurious,'
'harmless' or 'nontoxic to humans and pets'...." Historical inter-
pretation of this section has prohibited label statements concern-
ing the lack of toxicity or effect to specific predators and para-
sites. The Agency could amend this section to allow claims as to
lack of adverse effect on beneficial agents critical to IPM and
crop production systems when supported by the appropriate data.
(It is not the intent of this proposal to sanction any claims as
to the safety to or lack of effect on humans, pets, or the environ-
ment, since such claims are not considered to contribute signif-
icantly to the success of programs relating to IPM. The lower
degree of risk inherent in biorational pesticides will be discerned
by the label signal words and the relative reduction of precau-
tionary statements.) This amendment would be only for those agents
subject to Subdivision M guidelines. Data in support of these
claims would be submitted under Subdivision M guidelines, section
series 154, on Non-Target Organism Hazards.
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SUBDIVISION H — GUIDELINES FOR BIORATXONAL PESTICIDES
S«ri«S 150: OVERVIEW, DEFINITIONS, AMD GENERAL PROVISIONS
§ 150-1 Overview.
(a) Scope and purpose. This subdivision describes the kinds
of data required by 40 CFR Part 158 to support the registration of
biorational pesticide products. Biorational pesticides considered
in these guidelines include both biochemical pest control agents
(hereinafter called "biochemical agents") and microbial pest control
agents (hereinafter called "microbial agents"). Pesticides that
do not aeet the definition of "biorational pesticide" (i.e.,
biochemical or microbial agents) as set forth in 40 CFR § 158.65
shall be tested as required by the other sections of Part 158.
Certain biological control agents (e.g., macroscopic predators
and parasites) are exempt from the requirements of FIFRA, as
authorized by sec. 25(b) of FIFRA and specified in the Exemption
from Regulation of Certain Biological Control Agents published in
the Federal Register of June 2, 1982 (47 FR 23928). Generally,
the testing guidelines for biochemical agents are delineated
separately in this subdivision from those for microbial agents.
Each section in this subdivision identifies the kinds of data
required by Part 158, the standards that the studies and data
should meet, and the conditions under which each )cind of data is
required as specified in Part 158.
(b) Exceptions to testing biorationals under Subpart M.
Although certain pesticides may be biorational pesticides by defini-
tion, it may be more appropriate to test them as conventional pes-
ticides (for biochemicals) or to require testing at higher tiers
e.g., Tier II or III) as part of the minimum testing requirements
(for microbials). The following are some examples of characteris-
tics of pesticides that may indicate that the pesticide should not
be tested as an ordinary biorational pesticide:
(1) The active ingredient(s) or any of its (their) metabolites,
degradation products, or impurities is (are) structurally related
to a recognized carcinogen, and a theoretical vorst'omse risk based
upon the dose-response relationship for the most potent tested
animal carcinogen in the chemical class exceeds the. jri*k permitted
for nitrosamines and other unintentional contaminant* of pesticide
products.
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(2) The product is a genetically engineered microorganism
with characteristic* or species relationships closely allied to
known pathogens that cause serious crop damage, human infections,
serious diseases of domestic animals, or similar problems.
(c) Relationship to other subdivisions of the guidelines.
This subdivision constitutes a complete set of guidelines for
biorational pesticides. To avoid needless duplication, references
are made to other subdivisions whenever the guidelines axe iden-
tical. Each section series In this subdivision corresponds to a
subdivision of the pesticide guidelines for conventional pesti-
cides.
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 Label Development corresponds to Subdivision H —
Labeling for Pesticides and Devices.
$ 150-2 Definitions.
(a) Terns used in this subdivision are defined in FIFRA, in
§ 162.3 of the FZFRA sec. 3 regulations, and in the following
sections of the guidelines:
§ 60-2 of Subdivision D
§ 70-2 of Subdivision S
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§ 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) "Adjusted host equivalent" means the host equivalent
times the ratio of the weight of the test organisms to the weight
of the host organism.
(2) "Xllomone" means a chemical emitted by one species that
modifies the behavior of a different species to the benefit of the
emitting species.
(3) "Aquatic animal*" means all vertebrates and invertebrates
that inhabit fresh, estuarine, or marine waters for all or part of
their life cycles.
(4) "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.
(5) "Biochemical pest control agent" means a semlochemical,
plant regulator, hormone, or enzyme used as a pesticide.
(6) "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.
„ (7) "Biorational pesticide" means microblal pest control
agents such as viruses, bacteria/ protozoa, fungi and biochemical
pest control agents, either naturally occurring or identical to a
natural product, that attract, retard, destroy, or otherwise exert
a pesticidal activity. The Agency will determine on a case-by-case
basis whether synthetic biochemical agents not identical to natural
biochemical agents are biorational pesticides.
(8) "Environmental expression" means the extent and manner in
which a microorganism establishes and maintains its presence in an
ecological niche.
(9) "Inzootic" means a disease that is present in an animal
population at all times but that occurs in only small numbers of
individuals at any given time.
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(10) "Epizootic" means a disease attacking many animals in a
population at the same time; widely diffused and rapidly spreading.
(11) "Estimated environmental concentration" means an estimate
of the concentration of a biorational pesticide occurring in or on
various media (i.e., soil, water/ air) after pesticide application,
as determined from the results of environmental fate Tier II testing
(H 155-4 through -13) or environmental expression Tier II testing
($$ 155-18 through -20).
(12) "Genetic engineering" means to artificially alter the
genetic constitution of an organism by recombinant OKA techniques.
(13) "Habitat" means the plants, animals, and physical
components of the environment that constitute the natural food,
physical-chemical conditions, and cover requirements of an organism.
(14) "High volatility" means that the estimated volatility of
a substance based on vapor pressure and solubility in water is
greater than 5 x 10*5 »tm. m.3/mole ((derived from Henry's Law
Constant H • vapor pressure (atm)/ water solubility (gmole pesti-
cide/m3 water].
(15) "Hormone" means a chemical agent, produced by a tissue
or endocrine gland, that controls physiological functions or behavior
of an organism.
(16) "Host equivalent" means that amount of microbial agent
active ingredient that one infected host (e.g., a late instar larva)
can contain.
(17) "Hydrosoil" means the sediment underlying bodies of
water.
(18) "Improved strain" means an altered organism of potentially
increased benefit to man created by causing a small change in the
make up or sequence of the genetic material by chemical, radiation
or other external mutation means.
(19) "Kairomone" means a chemical emitted by one species that
modifies the behavior of a different species to the benefit of the
receptor species.
(20) "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.
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(21) "Maximum hazard testing* means testing that is designed
to maximize the test (non-target) organism's susceptibility to any
toxic or pathogenic effects of the test substance.
(22) "Microbial pest control agent" naana any of those
bacteria, viruses, protozoa, and fungi that are used to control
pests. Other aicrobial entities will be considered to be microbial
pest control agents as they are developed.
(23) "Natural occurrence" means the occurrence of an organism
in its normal niche where it grows, develops, and reproduces.
(24) "Naturally occurring" means the natural concentration or
population of the organism or biochemical (that could also be
employed as a pest control agent) present as airborne, particulate,
or liquid material and that would be in a form that could result in
exposure of nontarget organisms, including humans.
(25) "Niche" means the ecological position or function of an
organism in a community of plants and animals.
(26) "opportunistic pathogen" means a microorganism that
taxes advantage of a temporary environmental or host condition
(i.e., host disability or weakness) to cause a disease in the host
organism.
(27) "Passive dispensing device" means an apparatus (e.g.,
hollow fiber container, impregnated substrate) used to dispense a
pesticide into the air through volatilization.
(28) "Pheromone" means a chemical substance produced by an
organism (e.g., insects) that modifies the behavior of other
individuals of the same species.
(29) "Plant regulator (natural)" means a substance produced
by a plant that alters plant growth, development, and differentation.
(30) "Purest infective form* means that preparation of
infective virus containing the least amount of extraneous material.
(31) "Semiochemical* means a chemical emitted by a plant or
animal that modifies the behavior of receptor organisms of like or
different species; semiochemicals include pheromones, allomones,
and icairooones.
(32) Tor the purposes of section series 155 the term "substrate"
means the natural material in the environment to which the microbial
pest control agent is applied.
(33) "Terrestrial wildlife" means non-domestic birds or
animals.
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(34) "Toxin" means a poisonous substance, generated by a
microorganism, plant, or animal, capable of causing toxicosis when
introduced into body tissues but also capable of inducing a
counteragent or antitoxin.
(35) 'Typical end-use product* means a pesticide product
representative of a major formulation category (e.g., •mulsifiable
concentrate, granular product, wettable powder) that contains the
active ingredient of the registration applicant's product.
150-3 General provision*.
(a) Scope. The standards contained in this section apply to
all studies in this subdivision unless another section of this
subdivision contains a specific standard on the saae subject.
(b) Basic standards for testing. (1) Test substance for
biological and environmental studies, (i) Tests requiring use of
the technical grade of the active ingredient shall be conducted
with the manufacturing-use product if both are identical, or with
the technical grade of the active ingredient used to produce the
manufacturing-use or end-use pesticide product if not identical.
(ii) The lot of the substance tested should be the sane
throughout the duration of the study, and the research 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 should be selected to be as nearly identical to the
original lot as practical. Chemical or biological assays should be
performed to ensure composition identity and consistency.
(iii) Each lot of the test substance should be analyzed, to
the limits of technical feasibility, and the name and quantities of
ingredients, contaminants, and impurities listed. The determination
should include the quantity of unknown material, if any, so that 100
percent of the test sample is accounted for. The test substance
should be within the limits of parity, if any, a«xttfi«d in accordance
with H 151-15 or -25 of this subdivision.
(iv) if the test or control substance is to "be incorporated
into feed or other vehicle, the period "Soring which the test or
control substance is stable" in such a mixture should be determined
prior to the start of the study. Ho mixture of test or control
substance with the feed or vehicle should be maintained or used
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during a period exceeding the known stability of the test or con-
trol substance in the mixture. Alternatively, determinations of
the stability of the test or control substance in random samples
of the diet or vehicle mixture should 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 contained in
the mixture.
(v) If the test or control substance is incorporated into
feed or other vehicle, its homogeneity and concentration in the
diet should be determined prior to the start of the study and each
time a new mixture is prepared. Random sample* of the mixture should
be analyzed at least monthly to ensure that proper mixing, formu-
lation, 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 specified
in this subdivision, the Agency aay require, after consultation
with the applicant, data derived from testing to be conducted
with:
(A) An analytically or microbiologically [e.g., purest infec-
tive fora, (PIF) for viruses] pure grade of an active ingredient;
(B) The technical grade of an active ingredient;
(C) The labile form of infectious material (e.g., non-occluded
virus);
(D) An inert ingredient of a pesticide formulation;
(£) A contaminant or impurity;
(F) A metabolite (from animals or plants) or degradation
product of an active or inert ingredient;
(6) The end-use formulated product;
(H) Any additional substance (including other pesticides recom-
mended for tank-mixing with the test substance) thit enhances the
toxic activity of the product for which registration is sought; or
(I) .Any combination of the substances mentioned in paragraphs
(b)(D(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
should be selected so as to maintain accuracy of the dosage or
treatment.
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(11) A vehicle used to dissolve or-dilute the test substance
or positive control substance should be chosen to possess the
following characteristics if possible:
(A) Zt does not alter the absorption, distribution, metabolism,
or retention of the test substance;
(B) Zt does not alter the chemical or biological properties
of the test substance or enhance, reduce, or alter the toxic or
infective characteristics of the test substance;
(C) At the levels used in the study, it does not produce
physiological effects and Is nontoxic; and
(0) Zt closely resembles the vehicle, if any, to be used
under expected conditions of use of the pesticide product. Zt
should be identical to the vehicle if possible.
(3) Controls for biological and environmental studies.
Controls are used in the biological or environmental studies
contained in this subdivision to ensure that observed effects are
associated with the test substance exposure. The appropriate
control groups should be identical In every respect to the treated
groups except for exposure to the test substance.. Zn studies
involving animals or plants, all controls should (to the extent
possible) be from the same sourcet be of the same age, receive the
same care, and receive the same nutriment as the animals or plants
receiving the test substance* To prevent bias, a system to randomly
assign organisms (or groups) to treatment and control categories
is recommended when use of such a system is possible and appropriate
(double-blind study).
(i) Untreated (negative) controls. Untreated (negative)
control groups are usually recommended. Untreated controls receive
neither the test substance nor any ancillary material (vehicle).
(11) Vehicle control groups. (A) Zf a vehicle is used to
administer the test substance, a concurrent vehicle control group
may be recommended. 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 recommended.
(B) As provided in paragraph (b) OXlif^'ttls section, the
vehicle should be selected on the basis of information establishing
that it is son-toxic at the levels used in the study, has no indepen-
dent physiological effects, and does not alter the chemistry or
toxic!ty of the test substance. Zf, however, there are insufficient
data on the effects of the vehicle, testing of the vehicle may be
necessary.
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(iii-} Positive controls. Positive control* generally are not
necessary.. These serve as internal quality controls, and demonstrate
icnown 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 saae known or standard toxicant or infective agent. Consult
individual sections of this subdivision for those tests where a
positive control is recommended.
(iv) Historical controls. Historical control data are
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.
$ 150-4 Reporting of data.
Each test report submitted under this subdivision should satisfy
the reporting provisions of this section, unless a specific section
elsewhere in this subdivision directs otherwise.
(a) General requirements. (1) Identification. Each test
should identify:
(i) The name and address of the laboratory or site where the
test was performed; and
(ii) Each party primarily responsible for any written or other
matter contained in the report, and the portions of the report for
which the party is responsible.
(2) Verification. Each test report should be:
(i) Signed by each of the senior scientific personnel,
including the laboratory director responsible for performing and
supervising the testing and preparing, reviewing, and approving the
test report; and
(ii) Certified by the applicant or an authorised 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 should include all
information necessary to provide a complete and accurate description
ar.d evaluation of the test procedures and results. A test report
should contain at least three parts: a summary and evaluation of the
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test results, a description of the test procedures, and a listing
of the. data and information required by each applicable section of
this subdivision. Metric units of measurement must be used although
English units may be included. The systems may not be mixed (e.g.,
mg/sq. in.).
(1) Summary and evaluation of test results. This section of
the test report should contain a summary and analysis of the data,
and the conclusions drawn from the analysis. The summary should
highlight any and all positive results or observations, and any
deviations from control data values indicative of toxic effects.
The summary should be presented in sufficient detail to permit
independent evaluation of the results.
(2) Description of the test procedure. This section of the
test report should contain a full description of the test procedure.
If an applicant believes any of the reporting requirements are
inapplicable, he should submit an explanatory statement to this
effect. A full description of the test procedure should include but
not be limited to:
(i) Deviation from standards. The report should indicate all
ways in which the test procedure fails to meet applicable standards
for acceptable testing contained in this subdivision, and should
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, should be stated.
(iii) Substance tested. Identification of the test substance
should be provided, including:
(A) If the test substance is a chemical: chemical name,
molecular structure, and a qualitative and quantitative determina-
tion of its chemical composition (including names and quantities
of contaminants and impurities, within technically feasible limits.
The determination should also include quantities of unknown materials,
if any, to account for 100 percent of the sample;
(1) If the test substance is primarily Moae^iffalt scientific
name and, to the extent possible, serotype and strain or other appro-
priate 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 determination should also include quantities
of unknown materials, if any, to account for 100 percent of the
sample;
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(C) Manufacturer and lot number of the test substance, and
relevant properties of the substance tested, (i.e., physical state,
pH, stability, and purity); and
(D) Identification and composition of any vehicles (e.g.,
diluents, suspending agents, and eaulsifiers) or other materials
used in administering the test substance.
(iv) Animal and plant data. Animal and plant data should
include:
(A) Species and strain used, reasons for selection of species
(if the species is other than the species recommended by sections
of this subdivision), and reasons for selection of strain;
(B) Source of supply of test organisms;
(C) Description of any pretest conditioning; (D) Method
used to randomly assign animals or plants to test or
control groups;
(E) Numbers of animals of each sex in each test or control
group; and
(F) Age and condition of animals or plants at beginning of
study.
(v) environmental conditions. A description of the environ-
mental conditions under which the testing was conducted should be
reported. Further details may be provided by specific testing
sections .elsewhere in this subdivision.
(vi) Treatments or doses. For studies where test substance
applications, treatments, or dosings are made, a complete descrip-
tion of such should be reported. Further details may be provided
by specific testing sections elsewhere in this subdivision.
(vii) Treatment for diseases not caused by the test substance.
In studies 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 should reported. Buch description
should include:
(A) Identification of the test organisms affected and the
disease organism involved;
(B) The nature and severity of the disease;
(C) The date of first observation and duration of the disease;
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(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, and the name and address
of the individual who is responsible for the archives, should be
reported.
(x) References. References should 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, maxing 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.
(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
should reflect the current state of the art.
(2) Standard deviation and standard error. All data averages
or means should 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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Series 151: PRODUCT ANALYSIS GUIDELINES FOR BIORATIONAL PESTICIDES
§ 151-1 General Information.
(a) Scop* ef product analysis requirements. (1) Thi*
••ction series outlines guidelines for the submittal of data and
information on product analysis la support of applications for
registration of both biochemical pest control agents and microbial
pest control agents. These guidelines generally parallel those for
conventional chemical pesticide products specified in Subdivision D.
(2) Sections 151-10 through - 18 refer to guidelines for
biochemical agents and H 151-20 through -26 refer to guidelines
for aicrobial pest control agents.
§ 151-2 through -9 [Reserved]
Subseries 151A: PRODUCT ANALYSIS GUIDELINES FOR BIOCHEMICAL PEST
CONTROL AGENTS
§ 151-10 Product identity and disclosure of ingredients.
(a) Product identity. As required by 40 CTR f 158.165, each
application for the registration of a biochemical pest control
agent that is a pesticide product shall contain the product name
and the trade naaa(s) (if different). The company code number(s)
may be given.
(b) Confidential statement of formula. As required by 40 CFR
§ 158.165, an application for registration of a product shall
contain a confidential statement of formula. 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 specifies certified
limits to be established should be listed. A separate confidential
statement of formula is required for each alternate formula of a
product. See FIFRA sec. 10 for requirements related to protection
of trade secrets.
(c) Information on ingredients. As required by 40 CTR
$ 158.165, an application for registration should contain the
following information (if available) on each ingredient which is
listed in the confidential statement of formula required by paragraph
(b) of this section:
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( 1) Each biochemical (including microbial toxins) should be
identified by:
(i) The chemical naae(s) from the Chemical Abstracts 1972-
1976 Index of Nomenclature, or other well-defined name;
(ii) The Chemical Abstracts (CAS) Registry tlumber(s);
(iii) The structural formula(s), empirical forvula(s);
(iv) The amount of biochemical present in the product in
recognized units of potency or other appropriate expression of
biological activity or percentage of weight;
(v) The genus and species names of the organism(s) from which
the biochemical was separated or with which it is commonly associated;
and
(vi) The specificity or host range of the biochemical activity
and mode of action. With respect to mode of action of the biochemical,
the applicant should discuss any potential hazard to man, the
environment, or non-target species.
(2) Ingredients, other than biochemical*, shall be identified
by:
(i) Percentage composition (by weight) of each ingredient;
(ii) Whether the ingredient is an active ingredient, an
intentionally added ingredient, or an impurity;
(iii) The chemical name from the Chemical Abstracts 1972-1976
Index of Nomenclature, or other well-defined name;
(iv) The Chemical Abstracts (CAS) Registry Number;
(v) The product name, the trade name, and the common name (if
established);
(vi) The experimental or internal code number;
(vii) For each active ingredient other than the biochemical,
the empirical formula, and the molecular weight or the molecular
weight range;
(viii) The structural formula, if it can be determined.
(3) The composition.limits shall be given for each ingredient
for which § 158.110 of 40 CFR Part 158 requires limits to be certified.
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If space permits, this information can be listed in the confidential
statement of formula; otherwise, a separate statement on certification
of limits must be submitted.
§ 151-11 Manufacturing process.
As required by 40 CTR $ 158.165, each product's registration
must be supported by an accurate and current description of the
process used to manufacture or formulate the product. The descrip-
tion shall contain the following information:
(a) Description of the basic manufacturing process. (1) For
each biochemical derived from biological sources:
(i) The starting material shall be listed;
(ii) The steps taken, both chemical and biological, to ensure
the integrity of the starting material and to limit the extraneous
contamination in the unformulated biochemical shall be given;
(iii) The procedures by which the manufacturer established
the identity and purity of the seed stock from which the unforaulated
biochemical is produced shall be described; and
(iv) The quality control methods and the techniques used to
ensure a uniform or standardized product shall be reported. Unless
the quality control methods are well established and recognized,
they shall be submitted in detail with information regarding their
accuracy, sensitivity, and interfering substances.
(2) For other ingredients, active and inert, the guidelines
are those set forth in § 61-2 of Subdivision 0.
(b) Toxic or sensitizing substances. If the presence of
ingredients toxic or sensitizing to humans or other nontarget
mammalian species is suspected at any stage of the manufacturing
process, then data must be submitted to show that the substances do
not exist in the final biochemical product or exist only in quantities
too small to pose any hazard.
§ 151-12 Discussion on the formation of unintentional ingredients.
As required by 40 CTR § 158.165, a registration application
shall include a discussion concerning potential formation and
presence of unintentional ingredients in the product in quantities
which may produce adverse human or environmental effects. As
described under § 61-3 of Subdivision D, such unintentional ingre-
dients may be introduced during th« manufacturing process vith the
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starting material, process solvents, equipment, packaging, and
other sources; from side reactions in the manufacturing process;
from interactions between ingredients; and from the degradation of
ingredients. The applicant should base his discussion on established
chemical theory. For biochemicala, the unintentional ingredients
can include but are not limited to extraneous host residues and
residues of contaminants that rea&in following the extraction or
purification process.
§ 151-13 Analysis of samples.
A report on the results of preliminary analysis are required by
40 CFR $ 158.165 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 regard-
ing the analysis of samples shall apply.
$ 151-14 [Reserved]
§ 151-15 Certification of ingredient limits.
As required by 40 CFR § 153.165, each registration must be
supported by a certification of ingredient limits. The guidelines
of § 62-2 of Subdivision 0 regarding the certification of limits
shall apply.
§ 151-16 Analytical methods for certified limits.
As required by 40 CFR § 158.165, information concerning analy-
tical methods to verify certified limits are required to support the
registration of each manufacturing-use product and each end-use
product. The guidelines of § 62-3 of Subdivision D regarding analy-
tical methods for certified limits shall apply.
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§ 151-17 Physical and chemical properties.
As required by 40 CFR Part 158, data on physical and chemical
properties are required to support the registration of each manufac-
turing-use product and each end-use product. See 40 CFR $ 158*50
and § 158.165 to determine whether these data must be submitted;
Section II-B of this subdivision contains additional discussion of
the femulators' exemption and who, as a general rule, is responsible
for submission of the required data.
&
The data pertaining to physical and chemical properties are
listed in Table 1. Sections 9
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Table 1—SUMMARY OF PHYSICAL AKD CHEMICAL DATA FOR BIOCHEMICAL
PEST CONTROL AGENTS
Test Substance
Technical or
purer grade
of the active Hanufacturing-
property
Color
Physical state
Odor
Melting Point
Boiling point
Density or specific
gravity
Solubility
Vapor Pressure
Octanol/vater
partition coeff.
PH
Stability
Flaanability
-flashpoint
-flame extension
Storage stability
Viscosity
ingredient use product
Yes
Yes
Yes
Yes (solids)
Yes (liquids)
Yes
Yes
Yes (pure fora)
Yes (for non-polar
orgmnics, pure
form)
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Mo
No
Yes
No
No
No
Yes
No
Yes
(combustible
No
Yes
Yes
End-use
product
Yes
Yes
Yes
No
No
Yes
No
No
No
Yes
No
Yes
liquids only)
Yes
(aerosols only)
Yes
Yes
Miscibility No
Corrosion characteristics No
(liquids only
Yes Yes
(emulsifiable liquids only)
Yes Yes
(when packaged in metal, plastic,
or paper containers)
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Subseries 151B: PRODUCT ANALYSIS GUIDELINES TOR MICROBIAL
PEST CONTROL AGENTS
§ 151-20 Product identity and disclosure of ingredients.
(a) Product identity. As required by 40 CFR $ 158.165, each
application for registration of a microbial pest control agent
•hall contain the following information: the product name; the
trade naae(s) (if different). The company code nunber(s) may be
given.
(b) Confidential statement of formula. As required by 40 CFR
$ 158.165, an application for registration of a product shall contain
a confidential statement of formula. This statement shall include
the nature and quantity of 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 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 § 158.165 to support eacch application for
registration. (1) The identification of bacteria, protozoa,
viruses, or fungi in the product shall (to the extent possible)
include the following:
(i) The taxonomic position, serotype, and 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;
(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 any unusual morphological, biochemical,
or resistance characteristic(s) of the organism if such characteris-
tic (s) are different from the classic description of the organism;
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(v) The amount of microbial agent present in the product in
recognized units of potency, percentage of weight, units of viability
or replication, or other appropriate expression of biological
activity; and
(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
potential hazard (such as infactivity) to man, the environment, or
nontarget species should be discussed.
(2) An application for registration shall contain the following
information on each ingredient, other than the microbial agent,
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;
(ii) Whether the ingredient is an active ingredient, an
intentionally added ingredient, or an impurity;
(iii) The chemical name from the Chemical Abstracts 1972-1976
Index of Nomenclature, or other well-defined name;
(iv) The Chemical Abstracts (CAS) Registry Number;
(v) The product name, the trade name, and the common name (if
established);
(vi) The experimental or internal code number;
(vii) For each active ingredient other than the microbial
agents, the empirical formula, and the molecular weight or the
molecular weight range;
(viii) The structural formula (when .known); and
(ix) The composition limits for each ingredient for which
5 62-2 of Subdivision D requires limits to be certified. It space
permits, this information can be listed on the confidential state-
ment of formula; otherwise, a separate statement on certification
of limits must be submitted.
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§ 151-21 Manufacturing process.
As required by 40 CFR § 158.165, each application for registra-
tion of a manufacturin^-us* product or end-use product should
contain a description of th« basic manufacturing process as required
in § 61-2 of Subdivision D. The starting and intermediate materials
should be listed together with the steps taken to ensure the inte-
grity of these materials, and the steps taken to limit the extra-
neous contamination, both chemical and biological, in the unformu-
lated microbial agent* This description shall include the procedures
used by the manufacturer to establish the identity and purity of
the culture from which the unformulated microbial agent is produced,
the method of manufacture, and techniques used to ensure a uniform
or standardized product. The integrity of the product as determined
by the most specific and sensitive chemical or serological test
must be demonstrated. If the test is not a recognized standard
test, a detailed description of the test together with information
regarding specificity, interfering substances, accuracy, and
sensitivity must be provided.
§ 151-22 Discussion on formation of unintentional ingredients.
As required by 40 C7R $ 158.165, each registration application
should include the following information:
(a) Theoretical discussion. The theoretical discussion con-
cerns 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: bacterial and fungal toxins, allergens,
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 manu-
facturing process; fermentation residues from the growth of bacteria
or fungi; extraneous host residues from viruses produced in cell cul-
tures, 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 microbial agent; if parity twithin reasonable
limits) cannot be achieved, then the means of controlling contami-
nant levels to an acceptable limit must be delineated.
(b) Toxic or sensitizing substances. If substances toxic or
sensitizing to humans or other non-target mammalian species are
known or suspected to be present at any stage of the manufacturing
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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 aniaal pathogens. Human or other nontarget
animal pathogens such as (but not limited to) Shigella, Salaonella,
and Vibrio aust not be present in the manufacturing-use product or
the end-use product.
§ 151-23 Analysis of samples.
A report on the results of preliminary analysis are required by
40 C7R J 158.165 to support the registration of each manufacturing-
use product and those 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 of the microbial agent may be
substituted for the chemical analysis.
§ 151-24 [Reserved]
§ 151-25 Certification of ingredient limits_.
As required by 40 C?R § 158.165, each registration must be
supported by a certification of ingredient limits. The guidelines
of §§ 62-2 and 62-3 of Subdivision D regarding certification of
limits and analytical methods, respectively, shall apply. The limits
for microbial agents should also be expressed in terms such as in-
ternational units of potency per milligram when these are determined
in serological or other appropriate tests.
§ 151-26 Physical and Chemical properties.
(a) When required. Data on physical and chemical properties
are required to support the registration of each manufacturing-use
product and each end-use product. See 40 CFR § 158.50 and $ 158.165
to determine whether these data must be submitted; Section ZI-B of
this subdivision contains additional discussion of the femulators'
exemption and who, as a general rule, is responsible for submission
of the required data.
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(b) Substances tested. Table 2 presents the relevant data
pertaining to physical and chemical properties for microbial agents.
Sections 63-1 through -21 of Subdivision 0 should be consulted regard-
ing the -conduct and the specific provisions of the tests.
§ 151-27 Submittal of samples.
When required by the Agency, as provided in 40 CTR $ 158.165,
the applicant shall submit a sample of the technical grade of the
active ingredient, manufactiiringuM product, or ind-use product.
When required by the Agency, the applicant shall submit a sample of
any additional substances in the product as listed in § 150-10(c) of
this subdivision. The samples should be sent to: Chief of Chemical
and Biological Investigations Branch, Benefits and Field Studies
Division, OPP/OPTS (TS-768), U.S. Environmental Protection Agency,
Building 402 ARC-East, Beltsville, MD 20705.
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Table 2—SUMMARY OP PHYSICAL AND CHEMICAL DATA FOR
MICROBIAL PEST CONTROL AGENTS
Test Substance
Property
Color
Physical state
Odor
Density or specific
gravity
Stability
Storage stability
Viscosity
Miscibility
Corrosion characteristics
Technical grade
of the active
ingredient
Yes
Yes
Yes
Yes
Yes
Yes
No
Manufacturing-
ose product
Yes
Yes
Yes
Yes
No
Yes
Yes
End-use
product
Yes
Yes
Yes
No
No
Yes
Yes
No
No
(liquids only)
Yes Yes
(eaulsifiable liquids only)
Yes Yes
(when packaged in metal, plastic
or paper containers)
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Series 152: TOXICOLOGY GUIDELINES FOR BIORATIONAL PESTICIDES
§ 152-1 General information.
(a) General. This section series sets forth guideline* for
testing to determine the potential for detrimental effects to humans
and domestic animals caused by biorational pesticides. The section
series is divided into:
(1) Guidelines for the evaluation of biochemical pest control
agents set forth in JJ 152-10 through -29; and
(2} Guidelines for the evaluation of microbi-al pest control
agents set forth in §§ 152-30 through -53.
(b) Biochemical agents. Testing of biochemical agents for pos-
sible effects on humans and domestic animals is performed in a tier
sequence. The potential for adverse effects can be ascertained by
acute toxicity, irritation and hypersensitivity tests, by short term
mutagenicity tests/ and by cellular immune response studies. When
detrimental effects are found in the first tier of tests/ additional
studies at the Tier IX and III levels will be required as provided
in 40 CTR § 158.165. The tier sequence and studies involved are
outlined in Table 3.
(c) Microbial agents. The testing of microbial agents for possible
effects on humans and domestic animals is performed in a tier sequence.
These tests consist of acute toxicity/infactivity studies, cellular immune
response studies, and irritation, hypersensitivity, virulence enhancement,
tissue culture, teratogenicity, mutagenicity, subchronic, and chronic
studies. Mot all studies pertain to each organism at each tier. The
general tier sequence and studies involved for microbial agents are
outlined in Table 4.
§§ 152-2 through -9 [Reserved]
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TABLE 3—SUMMARY OF TIER TESTS ON BIOCHEMICAL PEST CONTROL AGENTS
Test Description of Species Tested Test Substance1
TIER I
LPSO Determination
Oral
Dermal
Inhalation
Irritation
Ocular, primary
Dermal, primary
Hypersensitivity
Immediate
Non-immediate
Mutagenieity Tests
Cellular Immune
Response
Rat
Rat, mouse, or rabbit
Rat, mouse, rabbit, or guinea pig
Albino rabbit
Guinea pig or albino rabbit
Human experience during
product development
Hamster or albino guinea pig
Microbial organisms (see text)
House
TG, MP, EP
TG, MP, EP
TG, MP, EP
MP, EP
MP, EP
TG, HP, EP
MP, EP
TG, MP, EP
TG
TIER II2
Mutagenieity Tests Mammalian cell (see text)
Subchronlc Oral Mouse, rat. or dog
Subchronic Dermal
Subchronic
Inhalation
Cellular Immune
Response
Rabbit or guinea pig (species not tested
in primary Tier I test)
Rat
Mouse
Teratogenicity Test Two species from rat, mouse, hamster,
rabbit
Rat
Newly weaned mouse; newly weaned rat
TIER
Chronic Exposure
Oncogenlc Test
TG, MP, EP
TG
TG
TG
TG
TG
TG
TG
1. Abbreviations used: MP • manufacturing-use product; EP - end-use product;
TG • technical grade or representative technical grade of active ingredient.
2. Not all tests may be indicated for each biochemical pest control agent; the
appropriate tests will depend on the results of Tier 1 and/or Tier II
tests.
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TAHI.E 4—SUMMAHY OF TIER TESTH ON M1CKOH1AI. I'EST CONTROL AGENTS
Test Substance*
Test
ER I
LD5U Determination
Oral
Dermal
Inhalation
Inf activity
Intravenous
Intracerebral
Intracerebral
Intraperltoneal
Intraperitoneal
Irritation
Dermal, primary
Ocular, primary
Hyper sensitivity
Immediate
Non- immediate
Non- immediate
Cellular Immune
Response
Tissue Culture
Description of Species Tested
and Information Concerning Test Bacteria Fungi Virus Protozoa
Rat TG, EP, MP
Rat or mouse TG, EP, MP
Mouse, rabbit, or guinea pig TG, EP, MP
Newly weaned mouse and hamster TG2
Newborn mouse i newborn hamster
Mouse and rabbit
Mouse and rabbit
Mouse and one other species
Guinea pig or rabbit EP, MP
Rabbit EP, MP
Human experience during product
development TG, MP, EP
Hamster or guinea pig MP, BP
Hamster Or guinea pig MP, EP
Mice TG
Various cell lines
(See section on viral agents)
TG, EP, MP
TG, EP. MP
TG, EP, MP
Mb"* 2
EP, MP
EP, MP
TG, MP, EP
MP, EP
MP, EP
TG
TG, EP, MP
TG, EP, MP
TG, BP, MP
PIF2
PIF2
EP, MP
EP. MP
TG, MP, EP
MP, EP
MP, EP
TG
PIF
TG,
TG,
TG,
TG2
TG2
EP,
EP.
TG,
MP,
MP.
TG
EP, MP
El>, MP
EP, MP
MP
MP
MP, EP
BP
EP
1 Abbreviations used: EP =« end-use formulated producti TG = technical grade or representative
technical grade of active ingredient} PIF - purest Infective form) MP ** manufacturing-use product.
2 One half of the animals in the test shall be immunodepressed.
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TABLE 4—CONTINUED
Test
Description of S|x*cica Testr-d and
Information Concerning Test
TIEM II3
Acute Oral
Acute Oral
Acute Inhalation
Acute Inhalation
Acute Interperitoneal
or Intracerebral
Subchronlc Oral
Primary Denial
Primary Ocular
Cellular Immune
Response
Teratogenlcity Test
Mutagenicity Tests
Virulence Enhancement
Puppies administered large doses
Newly weaned mousei newly weaned hamnter
A different species than used in Tier I
Newly weaned mousei newly weaned hamster
Two species other than those used in Tier If
Half the group are inmunodepreased.
Mice, rat, or dogi 90 day test
Guinea pigi use - dilution doses
Rabbit; use - dilution doses
Antibody formation cell mediated resjxjnse
Two species from rat, mouse, hamster, rabbit
Mammalian cell (see text)
Nice or hamster; serial passage
Teat Substance
Bacteria Fungi Virus Protozoa
TG
EP
EP
TG
TG
TG
TG
EP
EP
TG
TG
TG
MP, EP
NP, EP
EP
EP
TG
TG
TG.PIF
MP, EP
HP, EP
TG
TG
EP
EP
TG
TG
3 Not all tests may be indicated for each mlcroblal pest control agent; the appropriate tests will depend on the
results of Tier I and/or Tier II tests*
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TAIH.E 4—CONTINUED
Description of S|
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Subseries 152A: TOXICOLOGY DATA GUIDELINES TOR BIOCHEMICAL AGENTS
Group A-1: Tier I Tasting
§ 152-10 Acute oral toxicity study; Tier I.
(*) When required. Data from the acute oral L050 tests are
required by 40 CFR § 158.165 to support the registration of each
Manufacturing use product and each end-use product unless the
substance to be tested under paragraph (b) of this section is a
gas or highly volatile substance that cannot be administered
orally.
(b) Test standards. The test standards set forth in § 150-3
of this subdivision and $ fll-l(d) through (g) of Subdivision F
should be met, with the following exception:
(1) Route of administration. Intubation is the preferred
method of administering the oral dose.
(e) Reporting. In addition to the information required by
§ 150-4 of this subdivision and § 81(h) of Subdivision F, the
following should be reported:
(1) Information on gross pathology of animal tissues, organs,
and fluids, with emphasis on the gastro-intestinal tract as this
receives the initial challenge dose;
(2) Clinical signs of illness and toxicity such as elevated
temperature, unkempt appearance, altered feeding habits, weight
loss, and other signs of distress or physical depression; and
(3) Any signs of recovery from these symptoms.
(d) Tier progression. (1) If acute adverse effects (e.g.,
the LD50 is greater than 5 g/kg) axe observed, then:
(i) The subchronic oral dosing test ($ 152*20) shall be re-
quired as specified in 40 CFR $ 158.165 if either of the following
criteria is mett
(A) The use for which registration application is made re-
quires a tolerance for the pesticide or an exemption from the
requirement to obtain a tolerance, or requires the issuance of a
food additive regulationi or
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(B) The use of the pesticide product is likely to result in
repeated human exposure (e.g., from repeat applications or per-
sistence) to the product, its active ingredient(s), metabolite*s)
or degradation product(s) through the oral route; and
(ii) Teratogenicity studies ($ 152-23) shall be required
as specified in 40 CFR 158.165 if any of the following criteria
are net:
(A) Use of the pesticide, under widespread and commonly recog-
nized practice, may reasonably be expected to result in significant
exposure to human females; or
(B) Its use requires a tolerance or an exemption from the
requirement to obtain a tolerance, or its use requires issuance of
a food additive regulation.
(2) If no acute adverse effects are observed (e.g., the LD50
is greater than 5 gAg) / then no further testing is recommended.
§ 152-11 Acute dermal toxicity study: Tier I.
(a) When required. Data on the single-dose dermal 11)50 are
required by 40 CFR §158.165 to support the registration of each
manufacturing-use product and each enduse formulated product,
unless the substance to be tested under paragraph (b) of this
section is a gas or highly volatile substance that cannot be admi-
nistered dermally.
(b) Test standards. The test standards set forth in $ 150-3
of this subdivision and § 82-2(d) through (g) of Subdivision P should
be set with the following exceptions:
(1) Test species. A generally recognized strain of laboratory
rat, aouse, or rabbit should be tested.
(2) Number of animals and selection of dose levels, (i) A
trial test is recommended for the purpose of establishing a dosing
regimen which shall include one dose level higher than the expected
LDjQ and at least one dose level lower than the expected TLD50. If
data based on testing with at least 5 animals per sex with abraded
skin are submitted showing that the 11)50 *• greater ttoaa-2 g/kg for
the 24-hour contact period, no further testing at other dose levels
is necessary. If mortality occurs, the recommendations of paragraph
(b)(ii) of this section apply.
(ii) The number of animals per dose level, and the number and
spacing of dose levels should be chosen such that mortality rates
between 10 percent and 90 percent are produced, in order that
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119
calculation of the LD50 (abraded skin and intact skin) of males and
females with a 95 percent confidence interval of 20 percent or less
can be made. At least 3 dose levels of the test substance, in addition
to controls, should be tested; test groups shall contain approximately
equal numbers of male and female animals.
(c) Reporting. In addition to the information required by
§ 150-4, the following recommendations should be met:
(1) Information on the gross pathology of animal tissues,
organs, and fluids;
(2) Pathological changes to the skin receiving the initial
challenge dose;
(3) Clinical signs of illness or toxicity such as elevated
temperature, unkempt appearance, altered feeding habits, weight loss,
and other forms of distress or physical depression; and
(4) Any signs of recovery from these symptoms.
(d) Tier progression. (1) If acute adverse effects are
observed, then subchronic dermal toxicity tests (§ 152-21) shall be
required as specified in 40 CFR $ 156.165 when pesticide use is
likely to result in repeated human skin contact with the product,
its active ingredients, or their breakdown products.
(2) If no acute effects are observed (e.g., during testing,
greater than 2 g/kg), then no further testing is recommended.
§ 152-12 Acute inhalation toxieity study; Tier I.
(a) When required. (1) A determination of the acute inha-
lation toxicity is required by 40 CFR § 158.165 to support the regis-
tration of each manufacturing-use product if:
(i) The product is a gas;
(ii) The product produces a respirable vapor; or
(iii) Twenty percent or more of the aerodynamic equivalent of
the product is composed of particulates not larger .than 10 microns
in diameter.
(2) A determination of the acute inhalation toxicity is re-
quired by 40 era § 158.135 to support the registration of each end-use
formulated product if:
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(i) The end-use formulated product (as registered or under
conditions of use) is a gas, or causes a respirable vapor; or
(ii) Twenty percent or more of the aerodynamic equivalent of
the end-use product (as registered or under conditions of use) is
composed of particuLates not larger than 10 microns in diameter.
(b) Test standards. The test standards set forth in $ 150-3
of this subdivision and § 81-3(e-g) of Subdivision P should be met
with the following exception:
(1) Species. Testing should be performed with laboratory strains
of the rat, mouse, rabbit, or guinea pig.
(2) equipment. The particle size created by the disseminating
device must be in the respirable range for the species under test.
(c) Reporting. In addition to the information recommended by
§ 150-4 of this subdivision and § 81-3(h) of Subdivision P, the
following should be reported:
(1) Information on the gross pathology of animal tissues,
organs, and fluids;
(2) Clinical signs of illness or toxicity such as elevated
temperature, unkempt appearance, altered feeding habits, weight loss,
and other signs of distress and physical depression; and
(3) Any signs of recovery from these symptoms.
(d) Tier progression. (1) If acute effects are observed, then
the subchronic inhalation toxicity study (§ 158.135) would be
required as specified in 40 CPR § 158.165, when pesticide use may
result in repeated inhalation exposure at a concentration which is
likely to be toxic as determined from results of the acute inhalation
testing and other testing.
(2) 15 no acute effects are observed (e.g., the LC50 is greater
than 5 mg/liter for 4 hours duration), then no further testing is
recommended.
§ 152-13 Primary eye irritation study; Tier I.
(a) When required. (1) General requirement. Data on primary
eye irritation are required by 40 CFR § 158*165 to support the registra-
tion of each manufacturing-use product and each end-use product.
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121
(2) Corro«iv« pesticides. Data which demonstrate that the test
substance specified by paragraph (a) of this section has a pH of 1-
3 or 12-14 may b« submitted in lieu of data froo a primary eye
irritation study conducted in accordance with paragraph (b) of this
section. For all regulatory purposes, the Agency will assume that
such a substance is corrosive.
(3) Dermal irritation data. When studies conducted with the
test substance in accordance with § 152-14 indicate severe dermal
irritation, the data for that section will suffice to sect the
requirements of this section; the Agency will assume that a severe
dermal irritant will be a severe eye irritant.
(b) Test standards. The test standards set forth in § 150-3
of this subdivision and § 81-4(e & f) of Subdivision F should be met,
with the following exceptions:
(1) Test species. Testing should be performed on the albino
rabbit. Selection of other mammalian species may be acceptable, but
should be justified.
(c) Reporting. The reporting requirements set forth in § 150-4
of this subdivision and § 81-4(g) of Subdivision F should be met.
152-14 Primary dermal irritation study: Tier I.
(a) When required. (1) General requirement. Data on primary
dermal irritation are required by 40 CFR $ 158.165 to support the
registration of each manufacturing-use product and each end-use
product.
(2) Corrosive pesticides. Data which demonstrate that the test
substance specified by paragraph (a) of this section has a pH of 1-
3 or 12-14 may be submitted in lieu of data from a primary dermal
irritation study conducted in accordance with paragraph (b) of this
section. For all regulatory purposes, the Agency will assume that
such a substance is corrosive.
(b) Test standards. The test standards set forth in § 150-3
of this subdivision and § 81-5(e) and (f) of Subdivision F should be
met, with the following exceptions:
(1) Test species. Testing should be performed on either the
albino rabbit or the guinea pig. Selection of other mammalian species
may be acceptable, but should be justified.
(c) Reporting. The reporting requirements set forth in § 150-4
of this subdivision and § 81-5(g) of Subdivision F should be met.
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5 152-15 Hypersensitivity study; Tier I.
(a) Whan required. Data on hypersensitivity are required by
40 CTR § 158.165 to support the registration of each manufacturing-use
product and of «ach end-use prodduct whose us* will result in
repeated human skin contact under conditions of use.
(b) Test standards. The test standards set forth in § 150-3
of this subdivision and § 81-6(e) and (f) of Subdivision P shall be
met, with the following exceptions:
(1) Species. The test should be performed on at least one
mammalian species. The albino guinea pig and hamster are the preferred
species.
(2) Age and sex. Young adult males should be used when albino
guinea pigs are tested. Young adults of either sex may be used when
hamsters are tested.
(c) Reporting. The reporting requirements are the same as
those set forth in § 150-4 of this subdivision and § 81-€(g) of
Subdivision F.
§ 152-16 Bypersensitivity incidents; Tier I.
(a) When required. Data on incidents of hypersensitivity to
humans or domestic animals that occur during the production or testing
of the technical chemical, the manufacturing-use product, or end-use
product shall be reported as required by 40 CFR $ 158.165 with the
toxicology data supplied in support of an application for registra-
tion. For reporting of incidents taking place after registration,
refer to the requirements in sec. 6(a)(2) of PIFRA.
(b) Reporting. The reporting provisions for these incidents
shall be the same as those for conventional chemical pesticides, as
specified in the Pesticide Incident Report form (EPA form number 8550-
5, OMB number 158-R0008). The following infors*Uj9«unall be provided,
if available:
(1) The name of the biochemical agent)
(2) The length of exposure to the agent;
(3) The time, date, and location of exposure to the agent;
(4) The situation or circumstances under which exposure to
the agent occurred.
(5) Clinical observations.
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152-17 Studies to detect genotoxieity; Tier I.
(») When required. (1) Data derived from short-term microbial
mutagenicity tests are required by 40 C7R § 158.165 to support the
registration of each end-use product that meets any of the
criteria listed under (i)-(iii) below, and each manufacturing-use
product which may legally be used to formulate such an end-use
product. See 40 CFR 158.50 and 158.165 to determine whether these
data must be submittedt Section XI-B of this Subdivision contains
an additional discussion of the formulators' exemption and who, as
a general rule, is responsible for submission of the required
data.
(i) The use for which registration application is made requires
a tolerance or an exemption from the requirement to obtain a tolerance,
or requires issuance of a food additive regulation; or
(ii) The pesticide product is likely to result in significant
human exposure by inhalation or dermal routes before or during the
normal reproductive portion of the human lifespan; or
(iii) The active ingredient(s) or any of its (their) metabolites
is (are) structurally related to a known mutagen or oncogen, or be-
long(s) to any chemical class of compounds containing known mutagens
or oncogens. (Examples of chemical classes containing known mutagens
are alky la ting agents, N-nitroso-eompounds, polynuclear aromatic*,
heterocyclic hydrocarbons, certain natural products such as aflatoxins,
halogenated ethers and halohydrins, nucleic acid analogs, aromatic
amines, azo dyes, and nitro derivatives).
(b) General test standards. The applicable test standards set
forth in § 150-3 of this subdivision and § 84 of Subdivision F as
well as the following test standards apply to the conduct of all
studies necessary to produce the data outlined by this section:
(1) Test substance, (i) The technical grade of the active
ingredient or the manufacturing-use product shall be tested to
support the registration of a manufacturing-use product or an end-
use product.
(2) Tests to be performed. The mutagenicity tests should
include tests appropriate to address the following three categories:
(i) Oene mutations;
(ii) Structural chromosomal aberrations; and,
(iii) Other genotoxic effects as appropriate for the test
substance, e.g., numerical chromosome aberrations, direct UNA damage
and repair.
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Currently recognized tests for each of these categories are identified
m $ 84 of Subdivision F. Because of the rapid improvements in this
field, registrants are encouraged to discuss with the Agency: testing
battery selection, protocol design and results of preliminary testing.
(3) Replication. Ml tests should be repeated at least once for
reproducible determinations of response.
(4) Number and range of dose levels, (i) A suitable range of
concentrations should be used, including at least three concentrations
such that the lowest produces no effect (insignificant difference
from control) and the highest induces some toxicity (if possible) to
the test organisms.
(ii) For substances showing positive results, it is necessary
to obtain reproducible dose-response curves in a narrow range of
doses, if this is possible.
(5) Positive control groups. Concurrent positive control
substances should be selected for each test, in order to assure both
the sensitivity of the indicator organisms and the function of the
metabolic activation system.
(6) Negative control groups. Concurrent negative controls
should include the solvent, and, in addition, the test should include
either a concurrent non-solvent negative control or a historical
documentation for maintenance of genetic integrity of the indicator
organisms.
(c) Specific test standards for the tests. Each test should be
performed in accordance with the applicable standards described in
$ 84 of Subdivision F.
(d) Reporting. The reporting requirements set forth in § 150-4
of this subdivision should be followed.
(e) Tier progression. (1) If mutagenic effects are observed
in any microbial test, then short-term mammalian mutagenicity tests
(§ 152-19) shall be required as specified in 40 CFR § 156.165.
Consultation with the Agency is suggested to determine which tests
are to be performed, as this decision may be contingent upon which
microbial tests give positive results.
(2) It mutagenic effects are not obtained, as defined by the
standards of § 84 of Subdivision F, then no furtHeV testing is required
by 40 CFR $ 158.165.
(g) References. Refer to § 84-5 of Subdivision F.
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§ 152-18 Cellular immune response studies; Tier I.
(a) When required. Data on cellular immune response as deter-
mined from tests listed in paragraph (b) are required to support
the registration of each manufacturing-use product and each end-use
product. See 40 CFR $ 158.50 and § 158.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the formulators' exemption
and who, as a general rule/ is responsible for submission of the
required data.
(b) Test description. The following tests of effect(s) on
iamunocompetence are included in the cellular imstune response studies:
(1) Blood cell counts;
(2) Leukocyte number and cell classes, including T and B cells;
(3) Functional activity of blood leukocytes;
(4) Kacrophage number and function; and
(5) Serum protein determination.
(c) Test standards. In addition to the test standards set
forth in $ 150-3 of this subdivision, the following standards should
be followed:
(1) Test substance. The technical grade of each active ingre-
dient of the biochemical agent should be tested.
(2) Dosage. At least three dose levels of the biochemical
should be tested.
(3) Species. The test should be performed using appropriate
strains of the adult male and female laboratory mouse.
(4) Test methods. The following methods should be used for each
test:
(i) Blood cell count. Three groups of 10 male and female mice
each are separately injected (intraperitoneal) with 0.5 ml containing
a test substance undiluted, diluted lOx, or 4U4*4ifc£#0awvrespectively.
Three groups of 5 mice each similarly injected with physiological
saline should serve as controls. Routine blood counts should be per-
formed at 15 and 30 days after exposure. Standard hemocytometer
assays should be used to ascertain the total number of peripheral
blood leukocytes as well as differential counts.
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126
(ii) Leukocyte responses and T and B cell numbers. Three
groups of 10 male and female mice each are separately injected
(intraperitoneally) with 0.5 ml containing a test substance undi-
luted, diluted lOx, or diluted 10Ox, respectively. Three groups
of 5 nice each similarly treated with physiological saline should
serve as controls. Fifteen and 30 days after treatment/ treated
and control mice are to be tested for absolute peripheral blood T
and B lymphocyte numbers, and/or their ratio, in order to ascertain
whether or not there has been a significant shift in the populations
of these cells* For determining B cells, several standard assays
are available such as the immunofluorescent antibody test for
surface ZgG containing leukocytes. These tests should be used to
detect alterations in the ratio of peripheral blood leukocytes
having surface markers. Alternatively, sheep red blood cell rosette
assays with antibody sensitized erythrocytes or similar indirect
assays for cells with receptors for immunoglobulins could be used.
For the T cell, indirect immunofluorescent assays using standard
techniques should also be used. For example, antisera with reactivity
to specific mouse lymphocyte surface antigens (theta) will be used
in a standard fluorescent antibody test.
(iii) Functional activity. Functional activity of blood
leukocytes from test animals should be determined in regard to
responsiveness to plant mitogens such as phytohemagglutinin and/or
concanavalin A as indicators of T cell activity, and to B cell mitogens
such as endotoxln (bacterial lipopolysaccharide or dextran sulfate).
Give each of three groups of 10 male and female mice intraperitoneal
injections of 0.5 ml undiluted test substance, test substance diluted
10X, or test substance diluted 10OX, respectively. Three groups of
5 mice each similarly injected with physiological saline should serve
as controls. Control and treated mice should be tested at various
times thereafter, for example, at 15 and 30 days. After sacrifice,
peripheral leukocytes or splenocytes, and standard numbers of washed
cells (approximately 106 viable cells/culture) stimulated with at
least two doses of the above-named mitogens in triplicate should be
assessed for tritiated thymidine uptake by standard techniques. The
effect of the test substance on the expected leukocyte transformation
(i.e., blastogenesis) response of lymphoid cells to the mitogens
should be compared for control and test substance—treated animals to
determine whether there are any significant differences.
(iv) Macrophage number and function. Approximately 10 male or
female mice should be injected (Intraperitoneal) with 0.5 ml of
undiluted test substance. The number and percent of peritoneal and/or
splenic macrophages should be determined by stanttard phagocytic index
tests. Treated animals should be tested at least twice after exposure
(for example, at 15 and at 30 days) by:
(A) Determining the number and percent of peritoneal and splenic
macrophage uptake of latex particles, bacteria or yeast; or
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127
(B) Microscopic examination for percent of peritoneal and
splenic macrophages that adhere to glass plates; and
(C) The number of cultured adherent cells that show esterase
activity by means of histochemical stains.
(v) Serum protein determination. Approximately 10 male and
feaale mice should be injected (intraperitoneal) with 0.5 ml of
undiluted test biochemical. Seven and 15 days after exposure, serum
protein levels should be determined by standard radial gel-diffusion
assays and by electrophoresis to measure any overt effect(s) on such
levels. iBSBunoelectrophoresis can be used to measure effects on
concentrations of the different classes of ianunoglobulins found in
the gamma fraction of serum such as Ig G, A, M, and 0.
(d) Reporting. The reporting requirements set forth in § 150-
4 of this subdivision and § 80-4 of Subdivision F should be met.
(e) Tier progression. (1) If an indication of abnormality is
observed in any of the tests listed in paragraph-(b) of this section/
then the applicable Tier II cellular immune response studies (§ 152-24)
shall be required as specified in 40 CTR 158.165.
(2) If no abnormality is observed in any of the tests listed
in paragraph (b), then no further testing is recommended.
(f) References. The following references provide useful
information in developing acceptable protocols for cellular immune
response studies.
(1) Bloom, B.R., and J.R. David. 1976. In vitro Methods in Cell-
Mediated and Tumor Immunity. Academic Press, Inc., Mew York.
(2) Brunner, K.R., H.D. Engers, and J. Cerottini. 1976. The
Cr release assay as used for the quantitative measurement of cell-
mediated cytolysis in vitro. Pp. 423-428 in In vitro Methods in Cell-
Mediated and Tumor Immunity. B.R. Bloom and J.R. David, eds., Academic
Press, Inc., New York.
(3) Capel, P.J.A., W.P.M. Tamboer, R.M.ff. DeWaal, J.L.J. Jansen,
and R.A.P. Koene. 1979. Passive enhancement of souse skin all-grafts
by alloantibodies is Fc dependent. J. Imminol. 122:421-425.
(4) Fishbein, L. 1979. Studies in Kavirttesttiital Science 4:
Potential-Industrial Carcinogens and Mutagens. Klseiver Scientific
Publishing Co., New York.
(5) Garvey, J.S., N.E. Cramer, and D.H. Sussdorf. 1977.
Methods in Immunology. W.A. Benjamin, Inc., Reading, MA.
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128
(6) Harrington, J.R. 1974. Macrophage migration from an agarose
droplet: a micro-method for assay of delayed hypersensitivity in the
mouse. Cell. Immunol. 12:476-480.
(7) Jerne, N.K., and A. A. Nor din. 1963. Plaque formation in
agar by single antibody-producing cells. Science 140:405.
(8) Koski, I.R. , D.G. Pop lack, and R.M. Blaese. 1976. A
nonspecific esterase strain for the identification of monocytes and
macrophages . pp. 359-362 in In vitro Methods in Cell-Mediated and
Tumor Immunity. B.R. Bloom and J.R. David, eds. Academic Press,
Inc. , New York.
(9) Leijh, P.C.J., M.R. Van Den Barselaar, and R. Van Forth.
1977. Kenatics of phagocytosis and intracellular killing of Candida
albieans by human granulocytes and monocytes. Infect. I"miT 17:313-
318.
(10) Ling, N.R. , and J.C. Kay. 1975. Lymphocyte stimulation.
Elsevier, Amsterdam.
(11) Ma the, G. 1976. Cancer active iamunotherapy , Vol. 55.
Recent results in cancer research. Springer-Verlag, New York.
(12) Pepys, M.B., C. Sategna-Guidetti, and D.D. Marjah. 1976.
Enumeration of immunoglobulin-bearing lymphocytes in whole peripheral
blood. Clin. Exp. Tmmuno. 26:91-94.
(13) Rose, N.R. , and H. Friedman. 1976. Manual of Clinical
Immunology. American Society for Microbiology, Washington, D.C.
(14) Russell, E.S., and S.E. Bernstein. 1966. Blood and blood
formation, pp. 351-372 in Biology of the Laboratory Mouse. E.L.
Green (ed.), McGraw-Hill Book Company, New York.
(15) Schnyder, J., and M. Baggiollni. 1978. Role of phagocytosis
in the activation of macrophages. J . Exp . Med . 14:1449-1457.
(16) Territo, M.C. , D.W. Golde, and M.J. Cline. 1976. Macrophage
activation and function* Pp. 142-147 in Manual of Clinical Immunology.
N.R. Rose and H. Friedman, eds. American Society of Microbiology,
Washington, D.C.
(17) Werner, G.H., R. Karal, F. Floc'h, .fgdJb^Jouanna. 1977.
lexicological aspects of immunopotentiation by adjuvants and
immunostiaulating substances. Bulletin^ de L'Institut Pasteur 75: 5-
84.
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129
(18) Winchester, R.J.-, s.M. Fu, and T. Hoffman. 1975. IgG on
lymphocyte surfaces: technical problems and the significance of a
third cell population. J. Immunol. 114:1210-1212.
Group A-2: Tier II Testing
§ 152-19 Mammalian mutaqenieity tests; Tier II,
(a) When required: (1) Data from short-term mammalian
mutagenicity tests are required by 40 CTR 158.165 to support the
registration of each manufacturing-use product if positive results
were obtained in any one of the Tier I microbial tests (§ 152-17)
conducted to support registration of the manufacturing-use product.
(2) Data from short-term mammalian mutagenicity tests are
required to support the registration of each end-use product if
positive results were obtained in any one of the Tier I microbial
tests (§ 152-17) conducted to support registration of the end-use
product.
(3) Prior consultation with the Agency is suggested to
determine which of these tests must be performed. This would be
contingent upon which Tier I Microbial Test(s) gave positive results.
For example, if the Tier I test listed below in column "I" was
positive, then corresponding Tier II tests in column "II* would be
required as follows:
A. Positive Tier I Test B. Example of a Required Tier II Test
(A) Bacterial assay for ——> Mammalian cell gene mutation
(A) Bacterial assay for. ——> Mammalian cell gene mutation
reverse mutation (Ames) assay
(B) Prophage induction «——.-> Mammalian cell transformation
assay in lysogenic E. coli assay
(C) DNA damage/repair —--> In vivo mammalian cytogenetic
assay in E. eoli assay
(D) Yeast mitotic recom- ——> .In vitro mammalian cell cyto-
bination assay genetic assay
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130
See 40 CFR 158.50 and 156.165 to determine whether these data must be
submitted; Section II-B of this Subdivision contains an additional
discussion of the formulators' exemption and who, as a general rule, is
responsible for submission of the required data.
(b) General test standards. The general provisions for testing
as set forth in $ 150-3 of this subdivision, as well as the following
test standards, apply to the conduct of all studies necessary to
produce the data outlined in this section.
(1) Test substance, (i) The technical grade of the active
ingredient or the manufacturing-use product shall be tested.
(2) Tests to be performed. The required masnalian mutagenicity
assays may include tests appropriate to address one or more of the
following three categories depending on results of the Tier I
microbial assays:
(i) Gene mutations;
(ii) Structural chromosomal aberrations; and,
(iii) Other genotoxic effects as appropriate for the test
substance, e.g., numerical chromosome aberrations, direct DNA damage
and repair.
Currently recognized tests are specified in § 84 of Subdivision F.
Because of the rapid improvements in this field, registrants are
encouraged to discuss with the Agency: testing battery selection,
protocol design and results of Tier I testing.
(3) For substances showing positive results, it is necessary
to obtain reproducible dose-response curves in a narrow range of
dosages, if possible.
(4) Replication. Each of the tests selected according to the
criterion of paragraph (a)(1) above should be repeated at least once
for reproducible determination of response.
(5) Route of administration. For in vivo assays, the route of
administration should be that corresponding to potential human exposure.
(6) Positive control groups. Concurrent positive control
substances should be selected for each test in order to ensure the
sensitivity of the indicator organisms as well as"the function of the
metabolic -activation system for in vitro assays.
(7) Negative control groups. Concurrent negative controls
should include the solvent, and, in addition, the test should include
either a concurrent non-solvent negative control or a historical
documentation for maintenance of genetic integrity of the indicator
organisms.
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131
(c) Specific tests standards. Each test should be performed in
accordance with the applicable standards described in § 84 of
Subdivision F.
(d) Reporting. The applicable reporting requirements set forth
in § 150-4 of this subdivision and § 84 of Subdivision F should be
met.
(e) Tier progression. <1) If mutagenic effects (as defined
by positive results) are found in any one of the short-term mammalian
assays, then the Tier III oncogenicity studies (§ 152-29) are required
as specified by 40 CFR $ 158.165.
(2) If mutagenic effects are not obtained, then no further
testing is recommended*
(f) References. Refer to § 84-5 of Subdivision F. In addition,
the following references are provided for the mammalian cell
transformation assay.
(1) General;
(i) Bervald, Y., and L. Sachs. 1963. In vitro cell transfor-
mation with chemical carcinogens. Nature 200: 1182-1184.
(ii) Butterworth, B.E. 1979. Recommendations for practical
strategies for short-term testing for mutagens/carcinogens. Pp 89-
102 in strategies for Short-term Testing for Mutagens/Carcinogens.
3. Butterworth, ed. CRC Press, West Palm Beach, Fla.
(iii) Heidelberger, C. 1975. Chemical carcinogenesis. Ann.
Rev. Biochen. 44:79-121.
(iv) Krahn. D.F. 1979. The use of cultured mammalian cell
transformation systems to identify potential carcinogens. Pp 55-66
in Strategies for Short-term Testing for Mutagens/Carcinogens. B.
Butterworth, ed. CRC Press, West Palm Beach, Fla.
(2) HEC (Primary Syrian hamster embryo);
(i) Berwald, Y. and L. Sachs. 1965. In vitro transformation
of normal cells to tumor cells by carcinogenic hydrocarbons. J. Hat.
Cancer Inst. 35:641-661. (Original description of technique.)
(ii) Casto, B.C., N. Janosko, and J.A. DlfftOlo., 1977.
Development of a focus assay model for transformation of hamster
cells in vitro by chemical carcinogens. Cancer Res. 37:3508-3515.
(Detailed description of focus assay.)
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(iii) DiPaolo, J.A., P. Donovan, and R. Nelson. 1969.
Quantitative studies of _in vitro transformation by chemical carcino-
gens. J. Mat. Cancer Inst. 42:867-876. (Detailed description of
colony assay.)
(iv) Pienta, R., j. Poiley, and W. Lebherz. 1977. Morphological
transformation of early passage golden Syrian hamster embryo cells
derived froa cryo-preserved primary cultures as a reliable in vitro
bioassay for identifying diverse carcinogens. Int. J. Cancer 19:642-
655.
(v) Pienta, R., M. Shah, W. Lebherz, and A. Andrews. 1977.
Correlation of bacterial stutagenicity and hamster cell transformation
with tuBorigenicity induced by 2,4-toluenediamine. Cancer Lett. 3:45-
52.
(vi) Poiley, J., R. Pienta, and R. Raineri. 1976. Transforma-
tion of hamster embryo cells by N-2-acetylaminofluorene in the
presence of microsomal enzymes. NCI Careinogenesis Program, Fourth
Annual Collaborative Conference, Orlando, Florida, p. 85. U.S.
Dept. of Health, Education and Welfare, Public Health Service,
National Institutes of Health, Bethesda, Hd.
(vii) Omezawa, K., T. Hirakawa, M. Tanafca, Y. Katoh, and S.
TaJeayema. 1978. Statistical evaluation of Pienta's j.n vitro
carcinogenesis assay. Toxicol. Lett. 2:23-27.
(3) Ad-HEC (Adenovirus-infected Syrian hamster embryo);
(i) Hatch, G., P. Balwierz, B. Casto, and J. DiPaolo. 1978.
Characteristics of hamster cells transformed by the combined action
of chemical and virus. Int. J. Cancer 21:121-127.
(4) RLV-RE (Rauscher virua-infected Syrian hamster embryo);
(i) Dunkel, V., J. Wolff III, R. Pienta. 1978. In vitro
transformation as a presumptive test for detecting chemical carcinogens.
The Cancer Bull. 29:167-174.
(ii) Freeman, A., E. Weisburger, J. Weisburger, R. Wolford,
J. MaryaJt, and R. Huebner. Transformation of cell cultures as an
indication of the carcinogenic potential of chemicals. J. Mat. Cancer
Inst. 51:799-808.
(iii)' Mishra, N., C. Wilson, X. Pant, and P. Thomas. 1978.
Simultaneous determination of cellular smtagenesis and transformation
of chemical carcinogens in Fischer rat embryo cells. J. Toxicol. and
Environ. Health 4:79-91.
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(5) BHX-21 (Baby hamster kidney);
(i.) Ishii, Y., J.A. Elliot, N.K. Mishra, and M. Leiberman.
1977. Quantitative studies of transformation by chemical carcinogens
and ultraviolet light using * subclone of BfflCji Clone 13 Syrian
hamster cells. Cancer Res. 37:2023-2029.
(ii) Purchase, I.T.H., E. Longstaff, E. Ashby, J.A. Styles,
D.Anderson, P.A. Lefevre, and F.R. Westvood. 1978. An evaluation
of six shortterm tests for detecting organic chemical carcinogens.
Appendix III. Mammalian cell transformation. Brit. J. Cancer 37:931-
935.
(iii) Styles, J.A. 1977. A method for detecting carcinogenic
organic chemicals using mammalian cells in culture. Br. J. Cancer
36:558-563.
(6) C3H-3T3 (CH3 mouse embryo fibroblast);
(i) Kaxunaga, T. 1973. A quantitative system for assay of
malignant transformation by chemical carcinogens using a clone derived
from BALB/3T3. Int. J. Cancer 12:463-473.
(7) Balb-10T1/2 (Salb mouse embryo fibroblast);
(i) Reznikoff, C.A., D.W. Branxow, and C« Heidelberger. 1973.
Establishment and characterization of a cloned line of C3U mouse
embryo cells sensitive to poatconfluence inhibition of division,
Cancer Res. 33: 3231-3238. (Origin of C3H 10T1/2.)
(ii) Reznikoff, C.A., J.S. Barstram, O.K. Brankow, and C.
Heidelberger. 1973. Quantitative and qualitative studies of chemical
transformation of cloned C3H mouse embryo cells sensitive to
postconfluence inhibition of cell division. Cancer Res. 33:3239-
3249.
§ 152-20 Subchronic oral dosing studies; Tier II.
(a) When required. Data from subchronic oral dosing studies
are required by 40 CFR § 158.165 to support the registration of each
end-use product for which acute adverse effects were observed during
acute oral toxicicy studies (§ 152-10) and each •aoafaeturing-ose
product which may legally be used to formulate such an end-use
product when either of the criteria presented in (1) and (2) below,
are met. See 40 CFR $ 158.50 and-f-458.165 to determine-whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the f emulators' exemption
and who, as a general rule, is responsible for submission of the
required data.
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(1) The use for which regi»tration application is made requires
a tolerance for the pesticide or an exemption from the requirement
to obtain a tolerance/ or requires issuing a food additive regulation;
or
(2) The use of the pesticide product is likely to result in
repeated human exposure to the product, its active ingredient(s), or
degradation product(s) through the oral route.
(b) Test standards* The test standards are set forth in
$ 150-3 of this subdivision and $ 82-1(c) of Subdivision F.
(c) Reporting. The reporting provisions are the same as
those required in § 82-1(h) of Subdivision F.
(d) Tier progression. (1) Data on a chronic exposure study
(§ 152-26) are required by 40 CFR 158.165 if the potential for
adverse chronic effects are indicated based on:
{£) The subchronic effect level established in this study;
(ii) The pesticide use pattern (e.g., rate, frequency, and
location of application); and
(iii) The frequency and level of repeated human exposure that
is expected.
(2) Data on an oncogenicity study (§ 152-29) are required by
40 CFR 158.165 if the test results of this study reveal a morphologic
effect (e.g., hyperplasia, metaplasia) in any organ that potentially
could lead to neoplastic change.
(3) If the potential for chronic adverse effects is not indi-
cated by paragraph (d)(1)(i),(ii), and (iii) of this section, and
no morphological effects are noted (in any organ) that potentially
could lead to neoplastic change, then no additional testing is
recommended.
§ 152-21 Subehronie dermal toxicity study. Tier II
(a) When required* Data from the subchronic dermal toxicity
studies are required by 40 CFR \ 158.165 to support the registration
of each end-use product for which acute* adverse effects were observed
during acute dermal toxicity studies ($ 152-11) and each manufactur-
ing-use product which may legally be used to formulate such an
end-use product and when the pesticide use is likely to result
repeated human skin contact with the product, its active ingre-
dients , or its breakdown products. See 40 CFR § 158.50 and
§ 158.165 to determine whether these data must be submitted;
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Section IZ-B of this subdivision contains an additional discussion
of the formulatori' exemption and who, as a general rule, is respon-
sible for submission of the required data.
(b) Test standards. The test standards are set forth in § 150-3
of this subdivision and $ 82-2(b) of Subdivision F.
(c) Reporting. The reporting provisions are the same as
those for testing conventional chemical pesticides as set forth in
§ 82-2(h) of Subdivision F.
(d) Tier progression. (1) Data on a chronic exposure study
(f 152-26) are required by 40 CFR 156.165 if a potential for adverse
chronic effects is indicated, based on:
(i) The subchronic effect levels established in this study;
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(b) Teat standards. The test standards set forth in $ 150-3
of this subdivision and § 82-4(c) of Subdivision F should be met, with
the following exceptions:
(1) Equipment. The particle size created by the disseminating
device nust be in the respirable range for the species under test.
(2) (Reserved}
(c) Reporting* The reporting provisions are the same as
those set forth in § 82-4(g) of Subdivision F.
(d) Tier progression* (1) Data on a chronic exposure study
<$ 152-26) are required by 40 CTR § 158.165 if the potential for
adverse chronic effects is indicated, based on:
(i) The subchronic effect levels established in this study;
(ii) The pesticide use pattern (e.g., rate, frequency, and site
of application), and
(iii) The site, frequency, and level of repeated human exposure
that is expected.
(2) Data on an oncogenicity study (§ 152-29} are required
by 40 CFR $ 158.165 if the test results of this study reveal a morpho-
logic effect (e.g., hyperplasia, metaplasia) in any organ that
potentially could lead to neoplastic change.
(3) If the potential for chronic adverse effects is not indicated
based on paragraph (d)(1)(i), (ii)/ and (iii) of this section and no
morphologic effects are noted (in any organ) that potentially could
lead to neoplastic change, then no further testing is recommended.
§ 152-23 Teratogenicity studies; Tier II.
(a) When required. Data from teratogenicity studies are
required by 40 CFR $ 158*165 to support the registration of each
end-use product for which adverse effects were observed during
acute oral studies (§ 152-10) and each manufacturlne? use product
which nay legally be used to formulate such an end-use product
when either of the criteria in (1) or (2) below, are met. See 40
CFR § 158.50 and § 158.165 to determine whether these data must be
submitted; Section II-B of this subdivision contains an additional
discussion of the formulators' exemption and who, as a general rule,
is responsible for submission of the required data.
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(1) Use of the product under widespread and recognized practice
say reasonably be expected to result in significant exposure to female
humans; or
(2) its use requires a tolerance or an exemption from the
requirement for a tolerance, or its use requires issuance of a food
additive regulation.
(b) Test standards. The test standards are the same as those
set forth in $ 83-3(b) of Subdivision F.
(c) Reporting. The reporting requirements are the same as
those set forth in § 83-3(h) of Subdivision F.
§ 152-24 Cellular immune response studies: Tier II.
(*) When required. Data on cellular immune response studies
(Tier II) are required by 40 CFR $ 158.165 to support the registration
of each manufacturing-use product and each end-use product when
adverse effects are observed in the Tier I cellular immune response
studies described in § 152-18 of this subdivision.
(b) Test standards. In addition to the test standards set
forth in § 150-3 and § 152-18(c) of this subdivision, the following
standards should be met:
(1) Test substance. The technical grade of the active
ingredient(s) shall be tested.
(2) Species. Appropriate strains of the laboratory mouse should
be used to perform these studies.
(3) Test methods, (i) Antibody-forming activity. Immunized
mice are injected (intravenously) with the test substance and the
antibody response is subsequently assayed. Groups of at least five
mice each of a standard inbred strain are injected (intravenously)
with the maximum practical dose of the test substance. The animals
are then iosunized at two time intervals after treatment (for example
15 or 30 days) with a standardized dosesof an antigen such as sheep
erythrocytes (for example/ 4 x 106 washed red blood .cells). Four
days later/ the spleen is removed and the number?•("•picnic antibody
plaque-forming cells (PFC) in the spleen of animals exposed to various
levels of pesticides are to be determined. For this purpose the
standard hemolytic antibody plaque assay for B lymphocyte responses
to erythrocytes can be performed. It is not sufficient to merely
determine the serum antibody levels, since such tests are relatively
insensitive for detection of all but gross changes in antibody
responsiveness.
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(ii) Cell-mediated immune responses. To determine whether
cellaediated immune (CMI) responses have been effected by the test
substance, at least one assay for Oil should be performed either with
antigens derived from whole animals, from mammalian cells in culture,
or frost other antigens. The tests should be performed for animals
treated with the maximum doee of a test substance. A test should be
performed with groups of at least five animals each at two time
intervals after exposure, such as 15 or 30 days. Selection of the
specific assays are the option of the registration applicant. A
typical in vivo assay could be used to determine allogenic skin graft
rejection time and/or resistance of treated animals to highly allo-
genic tumor cells. For example/ groups of five mice each could be
given a full thickness allogenic skin graft, and mean survival
time determined in comparison to the survival time of allogenic
skin grafts on untreated animals. Alternatively, animals could be
injected with test tumor cells (e.g., mastacytoma or other well-
studied tumor cells) in which an LDSO can be readily established
for control animals. The effect of the pesticide on the resistance
ot susceptibility of the animals to challenge by the tumor cells
in terms of altering either the LDSO or the time of rejection of
the tumors could be assayed in comparison to controls. In vitro
assays could also be performed} for example, spleen cells from
ania&ls sensitized with a normal allogenic skin graft or given a
tumor cell injection could be assayed for quantitative cell-mediated
immune responses (CMI) followed by a standard chromium release
assay. For example, in a chromium- release assay chromium-labeled
target cells (of an appropriate donor strain) are exposed to sple-
nocytes from sensitized animals in vitro to ascertain responsiveness
of "killer" T lymphocytes present in treated animals. For such
tests, 10e lymphoid cells are obtained from sensitized animals,
either control animals, or animals treated with a maximum dose of
test substance at an earlier time (e.g., day 15). In addition,
lymphoid cells from treated animals could be tested for their
ability to generate the "migration inhibitory factor" in vitro.
For these tests, 106 splenocytes are placed in microcapillary tubes
with or without a specific antigen such as the purified protein frac-
tions of mycobacteria. Treated animals are first sensitized with
the Bacillus CamilleGuerin (BCG) or mycobacteria extract. The
ability of the mononuclear spleen cell suspension to migrate from
the chamber in the presence of antigen can be determined and used
as a correlate of cellular immunity. Many modifications of such
CMI responses isi vitro are available, including several well stan-
dardized migration inhibition type tests. In f«M<*-<*»«j a test for
blastogenic responsiveness of leukocytes similar"^^^Ss>'antigen
tests could be performed. For example, In a typical test system
spleen cells from treated animals are cultured in microwell chambers
with and without a mitotic stimulator such as phytohamaglutinin,
concanavalin A, lipopolysaccharide, or even an extract of allogenic
cells. The ability of the spleen cells to respond to these mitogens
jln vitro as measured by uptake of tritiated thymidine is considered
one assessment of cellular immunity.
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(c) Reporting. The reporting requirements are the same as
those set forth in $ 150-4 of this subdivision and § 80-4 of Sub-
division F.
(d) Tier progression. If adverse cellular effects suggesting
oncogenic potential are observed in this study, data from an onco-
genicity study (§ 152-29} are required by 40 CFR $ 158.165.
(e) References. Refer to $ 1S2-18(f).
§ 152-25 [Reserved]
Group A-3: Tier III Testing
§ 152-26 Chronic exposure study; Tier III.
(a) When required. Data on a chronic exposure study is re-
quired by 40 CFR $ 158.165 to support the registration of each end-use
product and each manufacturing-use product which may legally be
used to formulate such an end-use product if the potential for
adverse chronic effects are indicated based on:
(1} The subchronic effect levels established in the subchronic
oral toxicity studies (§ 152-20), the subchronic dermal toxicity
studies {§ 152-21), or the subchronic inhalation toxicity studies
($ 152-22);
(2) The pesticide use pattern (e.g., rate, frequency, and site
of application); and
(3) The frequency and level of repeated human exposure that is
expected.
(4) See 40 CFR f 158.50 and f 158.165 to determine whether
these data must be submitted; Section ZI-B of this subdivision
contains an additional discussion of the formulators' exemption
and who, as a general rule, is responsible for sulmission of the
required data.
(b) Combined testing. A, chronic feeding study may be combined
with an oncogenicity evaluation, pursuant to $ 152-29, provided that
standards for both types of testing are met.
(c) Test standards. The test standards set forth in $ 83-1(d)
and (e) of Subdivision F should be met with the exception of the
following:
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(1)• Route of administration. The route of administration should
be as similar as possible to the principal expected human exposure
route.
(i) Pesticides that need a tolerance or an exemption from the
requirement to obtain a tolerance or whose use requires a food ad-
ditive regulation should be administered in the diet (unless some
characteristic of the pesticide precludes dietary administration
to test animals).
(ii) In all cases, use of routes of administration not
corresponding .to the principal expected human exposure routes should
be justified in the test report. Factors such a*s absorption metabo-
lism, and distribution of the compound following administration as
well as results of previous tests should be taken into consideration
in selecting a route of administration other than that correspond-
ing to the principal expected human exposure route.
(iii) Specific test protocols for the inhalation or dermal
exposure route should be discussed with the Agency prior to initia-
tion of the test.
(d) Data reporting and evaluation. The reporting and evalua-
tion requirements are the same as those set forth in $ 83-1(f) of
Subdivision F.
§§ 152-27 through -28 [Reserved].
§ 152-29 Oncogenicity studies; Tier III.
(«) When required. Data from oncogenicity testing are re-
quired by 40 CFR § 158.165 to support the registration of each
end-use product and each manufacturing-use product which may legally
be used to formulate such an end-use product that meets either of
the following criteria:
(1) The active ingredient(s) or any of its (their) metabolites,
degradation products, or impurities produce(s) in subchronic studies
($§ 152-20, -21, or -22) a morphologic effect (e.g., hyperplasia,
metaplasia) in any organ that potentially could lead to neoplastic
change; or
(2) If adverse cellular effects suggesting oncogenic potential
are observed in cellular immune response studies ($ 152-24) or in
mammalian mutagenicity assays (§ 152-19).
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(b) Combined testing. An oncogenic evaluation may be combined
with a chronic feeding study, pursuant to § 152-26, provided that
standards for both types of testing are met.
(c) Test standards. The teat standards are the same as those
set forth in § 83-2(d) and (e) of Subdivision F.
(d) Reporting. The reporting provisions are the same as those
set forth in $ 83-2(f) of Subdivision ?.
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Subseries 152B: TOXICOLOGY DATA GUIDELINES FOR MICROSIAL PEST
CONTROL AGENTS
Group B-1: Tier I Testing.
152-30 Acute oral toxieity/inactivity study with mierobial pest
control agents: Ti«r I.
(a) When required. Acute oral toxicity/lnfactivity data are
required by 40 CTR $ 158.165 to support the registration of each
manufacturing-use product and each end-use product.
(b) Teat standards. In addition to the standards set forth
in § 150-3 of this subdivision and § 80-3 and § 81(d) through (g)
of Subdivision F, the following standards should be met:
(1) Species. Testing should be performed on the laboratory
rat.
(2) Number of animals and selection of dose levels.
(i) Trial testing is recommended to establish a dose level
greater than the LDSO. If submitted test data using at least five
animals per sex show that the oral LDSO is greater than 5 g/kg, no
further testing at other dose levels is necessary. If mortality
occurs, the provisions of paragraph (b)(2)(ii) of this section
apply.
(ii) At least 3 dose levels spaced appropriately should be
tested using adequate numbers of animals to form test groups with
mortality rates in the 10 to 90 percent range in order to permit
LDSO determinations for males and females with a 95 percent confi-
dence interval of 20 percent or less.
(iii) An adequate numbers of animals per dose level in addition
to those described in (b)(2)(ii) of this section should be exposed
to the mierobial agent so that 2 female rats and 2 male rats can
be sacrificed at 1 week post-treatment to examine tissues and
organs for gross pathology and presence of the viable mierobial
agent.
(iv) All animals should be dosed by gavage.
(3) Control animals* A concurrent group of animals treated with
the vehicle containing killed organisms (autoclaved) should be included
as a control in each acute oral LDSO study.
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(4) Dose quantification. Titers (of the microbial suspensions
administered to test aniaals) should be performed by plating dilutions
on laboratory surface media or other suitable media or on host
organisms to enumerate viable organisms.
(5) Duration of tests* Surviving exposed animals and controls
should be observed for 14 days or until all signs of reversible
infactivity or toxicity subside*, whichever occurs later.
(6) Conduct of test.
(i) Fasting. Food should be withheld from the animals during the
night prior to dosing.
(ii) Observation. The animals should be observed frequently during
the day of dosing and checked at least once each morning and late
afternoon thereafter. The following should be recorded even if the
animals recover completely from the exposure:
Nature and onset of all gross or visible clinical signs of
illness such as elevated temperature, unkempt appearance,
altered feeding habits, weight loss, various forms of dis-
tress, and physical depression.
(Ill) Examination of excreta. Urine and feces samples from the
test animals should be collected at 24, 48, and 72 hours following
test initiation and examined for the presence of the microbial
agent.
(iv) Assay for specific antibody production. If test duration
exceeds 14 days, than an assay for specific antibody production should
be performed.
(v) Sacrifice and necropsy. All test animals surviving at the
end of the observation period should be sacrificed. All test animals,
whether dying during the test or sacrificed, should be subjected to a
complete gross necropsy. In addition microorganism dissemination,
replication, and survival in animal tissues, organs, and fluids should
be determined, including survival in the intestinal tract. Samples
should be cultured on laboratory surface media or other suitable
media or host organisms to provide qualitative and quantitative
measurements of survival and multiplication of the microorganism.
(c) Data reporting and evaluation. la MUtt&tft.to the require-
ments In ( 80-4 of Subdivision r, the test report should include
the following information:
(1) Tabulation of response data by sex and dose level (i.e.,
number of animals dying per number of animals showing signs of
infectivity per number of animals exposed), and
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(i) Time of death after dosing;
(ii) The LD50, for each sex and test substance, calculated at
the end of the observation period (with method of calculation
specified) expressed in numbers of viable microorganisms per kg
body weight and mg test substance per kg body weight; and
(Hi) Dose-response carve and slope.
(2) In addition, gross pathology, microorganism dissemination,
replication, and survival in *"<"»«1 tissues, organs, and fluids should
be reported, including survival in the intestinal tract. Results of
the assay for specific antibody production should be reported, when
applicable.
(3) The test organism should be characterized according to genus,
species serotype and strain (according to current acceptable taxonomy),
and the percentage of unknown fermentation solids or other materials
present indicated to account for 100 percent of the sample.
(d) Tier progression.
(1). If evidence of infactivity, persistence, presence of viable
microbial agent in test animal excreta, replication, or toxic effects
are observed in the acute oral studies, then the following Tier II
testing shall be required as specified in 40 CTR $ 158.165:
(i) Bacteria or fungi. Acute intraperitoneal or intracerebral
tests shall be conducted in two animal species other than those
used in Tier I. Half of the test animals should be immunodepressed
(§ 152-43). The bacterial and fungal virulence enhancement study
(§ 152-48} shall be conducted.
(ii) Viruses. An acute oral infactivity study shall be conducted
on newly weaned mice and newly weaned hamsters ($ 152-40), and
the teratogenicity study (§ 152-47} shall be required.
(iii) Protozoa.
(A) An acute oral infactivity study in puppies should be con-
ducted using large doses of protozoa (f 152-40).
(B) A subchronic 90-day oral test in the mouse, rat, or dog -
should be performed (§ 152-42).
(2) If evidence of acute oral infectivity, organism persistence,
replication, or toxic properties (e.g., L050 greater than 5g/mg) is
not observed, additional testing will not be required.
(e) References*
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(1) The following references contain information useful for
developing an acceptable protocol.
(i) Fisher, R., and L. Rosner. 1959. Toxicology of the
microbial insecticide thuricide. J. Agric. Food Chen. 7:686-688.
(ii) Ford, S., and L. Friedman. 1967. Experimental study of
the pathogenicity of aspergilli for sd.ce. J. Bacteriol. 94:928-933.
(iii) Forsbarg, C.W. (ed.). 1976. Bacillus thuringiensis: its
effects on environmental quality. Publication no. 15385 of the
Environmental Secretariat, National Research Council of Canada.
{2} The following references provide information on acceptable
methods of calculating the LD50:
(i) Finney, D.J. 1971. Probit Analysis. 3rd Edition.
Chapters 3 and 4. Cambridge University Press. Cambridge, Eng.
(ii) Litchfield, J.T., Jr., and F. Wilcoxon. 1949. A simpli-
fied method of evaluating dose-effect experiments. J. Pharmacol.
Exp. Therap. 96:99-115.
(iii) Thompson, W.R. 1974. Dse of moving averages and inter-
polation to estimate median effective dose. Bacteriological Rev.
11:115-145.
(iv) Weil, C.S. 1952. Tables for convenient calculation of
median effective dose and instruction on their use. Biometrics
8:249-263.
§ 152-31 Acute dermal toxicity/infeetivity study with microbial
pest control agents; Tier I.
(a) When required. Data on acute dermal infactivity are
required by 40 CFR § 158.165 to support the registration of each
manufacturing-use and each end-use product.
(b) Test standards. In addition to the applicable standards
set forth in § 150-3 of this subdivision and $ 80-3 and § 81-2(d)
through (g) of Subdivision F, an acute dermal infectivity study
should meet the following standards:
(1) Species. Testing should be performed with at least one
mammalian species, preferably the rat or mouse.
(2) Sex and age. Young adult male and female animals should
be used.
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(3) Number of animals and selection of dose levels.
(i) A trial test is recommended for the purpose of establishing
a dosing regimen which should include one dose level higher than the
expected LD50. If data from abraded skin testa on at least 5 animals
of each sex are submitted showing that the dermal LD50 is greater than
2 g/kg for the 24-hour contact period, no further testing at other
dose levels is necessary* If Mortality is produced, the provisions
of paragraph (b)(3)(ii) of this section apply*
(ii) At least 3 dose levels spaced appropriately should be
tested using adequate numbers of animals to fora test groups with
mortality rates in the 10 to 90 percent range in, order to permit LD50
determinations (abraded skin and intact skin) for males and for females
with a 95 percent confidence interval of 20 percent or less. In addi-
tion, the requirements of paragraph (b)(3) (ill) of this section may
apply.
(iii) Data from tests performed with the use dilutions of a pro-
duct may be necessary if the use dilution is intended for application
as a mist or spray.
(4) Control an •'•"'Is. A concurrent untreated control group of
animals should be included in the test. A concurrent vehicle con-
trol group is recommended if a vehicle or diluent used in administering
the test substance is expected to elicit an important toxicologic
response, or if insufficient data exists on the acute effects of
the vehicle.
(5) Dose quantification. Titers of microbial suspensions to
test animals should be performed by plating dilutions on laboratory
surface or other suitable media or host organisms for enumeration
of viable organisms.
(6) Conduct of test.
(i) Application. In all animals, the application site should
be as free of hair as possible. In addition, the application sites
in abraded-skin groups should be abraded in such a way as to penetrate
the stratum corneum but not the dermis. The test substance must
be kept in contact with skin covering at least 10 percent of the
body surface for at least 24 hours. [See Draixe" T"W44) for equiva-
lent sq. cm. of body surface.] The preferred application site is
a band around the trunk of the test animal. „ A^fCmpping material
such as gauze covered by an impervious nonreactive rubberized or
plastic material should be used to retard evaporation and to keep
the test substance in contact with the skin. At the end of the
exposure period, the wrapping should be removed and the skin wiped
(but not washed) to remove remaining test substance.
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til) Duration of observation. Animals should be observed for at
least 14 days after dosing or until all signs of reversible infactiv-
ity or toxicity in survivors subside, whichever occurs later.
(iii) Observations. Th« animals should ba observed frequently
during the day of dosing and checked at laast once each morning
and late afternoon thereafter4 The following should ba recorded
even though the animals recover completely from the exposure:
nature and onset of all gross or visible clinical aigns of illness
such as elevated temperature, unkempt appearance, altered feeding
habits, weight loss, various forms of physical distress, depression,
and similar responses.
(iv) Assay for specific antibody production* If test dura-
tion exceeds 14 days, then an assay for antibody production should
be performed.
(v) Sacrifice and necropsy. All test and control animals sur-
viving at the end of the observation period (14 days) are
sacrificed. All test animals, whether dying during the test or
sacrificed, are subjected to a complete gross necropsy. In
addition to gross pathology, microorganism dissemination, replica-
tion, survival in animal tissue, organs, and fluids should be
determined, including survival in the skin. Samples should be
cultured on laboratory surface or other suitable media or host
organisms to provide qualitative and quantitative measurements of
survival and multiplication of the microorganism.
(vi) Bistopathology. Bcamination of skin should include histolo-
gical examination of treated tissues in accordance with § 80-3(b)(11)
of Subdivision F.
(c) Data reporting and evaluation. In addition to the appli-
cable general information required by § 80-4 of Subdivision F, the
test report should include the following information:
(1) Tabulation of response data by sex and dose level (number
of animals dying per number of animals showing signs of infectivity
per number of animals exposed);
(2) Time of death after dosing;
(3) Observations of signs and symptoms;
(4) Gross pathological findings;
(5) evidence of microorganism dissemination, replication, and
survival in animal tissues, organ*,•and fluids, including survival-
in skin;
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(60 LDSO determinations for each sex and for each test substance
for animals with abraded skin and for animals with intact skin cal-
culated at the end of the observation period (with method of calcu-
lation specified) expressed in numbers of viable microorganisms
per kg body weight and mg of test substance per kg body weight;
(7) 95 percent confidence interval for the LD50;
(8) Dose-response curve and slope; and
(9) Identification of the test microorganism, including:
(i) Genus/ species, serotype, and strain (to the extent possi-
ble), according to current acceptable taxonomy; and
(ii) Hie percent of unknown fermentation solids or other mater-
ials present to account for 100 percent of the sample.
(10) Results of assays for specific antibody production, when
applicable.
(d) Tier progression*
(1) No further testing is required by 40 CFR 158.165 for
viruses or protoxoa.
(2) If evidence of infectivity, organism persistence or repli-
cation, or toxic effects is observed following acute dermal studies
with bacteria or fungi, then acute intraperitoneal or intracerebral
tests ihall be conducted in two animal species other than those
used in Tier I (f 152-43) as specified by 40 CFR 156.165. Half of
the test animals should be immunodepressed.
(e) References»
(1) Draize, J.H., G. Woodward, and B.C. Calvary. 1944.
Methods for study of irritation and toxicity of substances applied
topically to skin and mucous membranes. J. Pharmacol. Exp. Ther.
83:377-390.
(2) Draize, J.H. 1965. Appraisal of the safety of chemicals
in foods, drugs and cosmetics - Dermal toxicity. Assoe. of Food
and Drug Officials of the United States. Topeka, Kansas. Pp.
46-59.
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$ 152-32 Acute inhalation toxicity/infeetivity study with mierebial
pest control agenrst Tier I.
(a) When required. Data on acute inhalation toxicity/infee-
tivity are required by 40 CFR $ 158.165 to support the registration
of each•manufacturing-use and each end-use product if 20 percent
or more of the aerodynamic equivalent of the product (as registered
or under conditions of use) is composed of particulates under 10
microns in diameter.
(b) Test standards. In addition to the applicable standards
set forth.in $ 150-3 of this subdivision and § 80-3 and $ 81-3{e)
through (g) Subdivision P, an acute inhalation LC50 study should
meet the following standards:
(1) Species. Testing should be performed with the laboratory
mouse/ rabbit/ or guinea pig.
(2) Sex and age. Young adult male and female animals should
be used.
(3) Number of animals and selection of dose levels.
(i) A trial test is recommended for the purpose of establish-
ing a dose regimen which should include one dose level higher than
the expected LCgg and at least one dose level below the expected
LCgQ. If data based on testing with at least 5 animals per sex
are submitted showing that the LCjo is greater than 5 mg. equivalent
of viable microbial agent for 4 hours duration, no further testing
at other dose levels is necessary. If death occurs, the require-
ments of paragraph (b)(3)(ii) of this section apply.
(ii) At least three dose levels should be chosen using an ade-
quate number of animals•to form test groups with mortality rates
between 10 percent and 90 percent, and to permit LCSO calculations
with a 95 percent confidence limit of 20 percent or less.
(4) Duration of test. In selecting the exposure period, allow-
ance must be made for chamber concentration equilibration time. If
no problems are encountered in maintaining a steady concentration
of the test substance in the chamber(s), the exposure period should
be at least 1 hour. If problems are encountered in maintaining a
steady concentration, the exposure period should last up to 4 hours.
The animals should be observed for 14 days, or until .signs of revers-
ible infectivity subside, whichever occurs later.
(5) Dose quantification. Titers of microbial suspensions ad-
ministered to test animals should be calculated by plating dilutions
on laboratory surface media or other suitable media or in host
organisms for enumeration of viable organisms.
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(6) Control groups.
(i) A concurrent untreated control group is necessary.
(ii) If any solvent, other than water, is used in generating
the exposure atmosphere, a vehicle control group is necessary.
The vehicle control group should be exposed to an atmosphere contain-
ing the greatest concentration of solvent present in any test
system.
(7) Exposure chamber design and operation.
(i) Inhalation exposure techniques described in this section
are based on the use of whole-body inhalation chambers that allow
experimental animals to receive whole-body dermal exposure and
possibly large oral exposure, as well as exposure by inhalation.
In some cases, the Investigators will wawnt to use other inhalation
exposure techniques involving face masks, head-only exposures,
intratracheal instillation, or other similar techniques that reduce
or preclude added dermal and oral exposures. Some alternative
techniques are described by Phalen (1976). When alternative tech-
niques are used, the procedures and results should be .reported in
a manner similar to that required with the use of whole-body inha-
lation chambers.
(ii) Animals should be tested in a dynamic air flow exposure
chamber. The chamber design should be chosen to enable production
of an evenly distributed exposure atmosphere throughout the chamber.
The chamber design should also minimize crowding of the test animals
and maximize their exposure to the test substance by the inhalation
route.
(8) Operation measurements. The following measurements should
be taken with care to avoid major fluctuations in air concentrations
or major discrepancies in the oepration of the chambers:
(i) Air flow. The rate of air flow through the chamber should
be measured continuously;
(ii) Chamber concentrations.
(A) nominal concentrations of organisms should" be calculated
for each run by dividing the amount of the test substance containing
known aombers of organisms per unit volume of the test substance
used for the generating system by the air flowing through the
chamber during the exposure.
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external exposure which is representative of the entire exposure
period. The system used to generate the aerosol should be such
that the chamber concentrations and particle size distributions are
controlled under stable conditions;, reflecting the current state-of-
the-art, and should not vary in a ran?* greater than 30 percent of
the average (range/mean equal to or less than 30 percent)}
(iii) Temperature and humidity* The temperature should be
maintained at 24 + 2» C, and the humidity within the chamber at
40-60 percent* Both should be monitored continuously;
(iv) Oxygen* The rate of air flow through the chamber should
be adjusted to insure that the oxygen content of the exposure atmos-
phere is at least 19 percent; and
(v) Particle site measurements* (A) General * The particle
size created by the disseminating device must be in the respirable
range for the species under test* • Aerosol particle sice measure-
ments should be made on samples taken at the breathing level of
the animals. These analyses should be carried out using techniques
and equipment reflective of the state-of-the-art. All of the
suspended aerosol {on a gravimetric basis) should be accounted
for/ even when most of theiaerosol is not respirable.
(B) Siting analysis. The sizing analysis should be in terms
of equivalent aerodynamic diameters and should be represented as
geometric mean (median) diameters and their geometric standard
deviations (see NIOSH syllabus in Appendix)/ as calculated from
log probability graphs or computer programs. The size analyses
should be carried out frequently during the development of the
generating system to ensure proper stability of aerosol particles,
and only as often thereafter during the exposure as is necessary
to determine adequately the consistency of particle distributions
to which the animals are exposed/ q»*int«JlnJl»g at least 20 percent
of the particles at 10 microns or less except when creation of par-
ticles in this size range would kill or injure the microorganism
under test. At a minimum, these analyses should be carried out
tvice per hour for liquid test substances/ and 4 times per hour
for dusts and powders.
(9) Observation. The animals should be observed frequently
during the day of dosing and checked at least once-each morning
and late afternoon for at least 14 days or until all signs of
reversible infectivity subside, whichever occurs later. The follow-
ing should be observed even though the animals recover completely
from the exposuret nature and onset of all gross or visible clinical
signs of illness such as elevated temperatures, unkempt appearance,
altered feeding habits, weight Iocs, various forma of distress and
depression, and similar expressions. In addition, an assay for
specific antibody production should be performed if test duration
exceeds 14 days.
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152
(10) Sacrifice and necropsy. All test animals surviving at
the end of the observation period should be sacrificed. All test
animals, whether dying during the test or sacrificed, should be
subjected to a complete gross necropsy. In addition, microorganism
dissemination, replication, and survival in animal tissues, organs,
and fluids, and survival in nasal passages, tracheae, bronchi, and
lungs should be determined. Samples should be cultured on laboratory
surface or other suitable media to provide qualitative and quantita-
tive measurements of survival in the host organism and multiplica-
tion of the test microbial.agent.
(c) Data reporting and evaluation. In addition to the
requirements in $ 80-4 of Subdivision F, the test report should
include the following data?
(1) Particulate sice;
(2) Description of the chamber design and operation, including
chamber, type, dimensions, source of make-up air and its conditioning
(heating or cooling) for use in the chamber, treatment of exhausted
air, housing and maintenance of the animals in the chambers, and
similar related information, equipment for measuring temperature
and humidity, the generating system, and the methods of analyzing
airborne concentrations of aicrobial agent and particle sizing
should be described;
(3) The following operation data should be tabulated'both in-
dividually and in summary form, using means and standard deviations
(with or without ranges) in tabular form. Ae data summaries should
be grouped according to experimental groups, and differences (such
as in temperature and airflow) should be tested for statistical
significance.
(i) Airflow rates 'through the chamber;
(ii) Chamber temperature and humidity;
(iii) Nominal concentrations of microbial agent;
(iv) Actual concentrations of microbial agent; and
(v) Median particle sizes and their geometric standard devia-
tions, and the percentage of particles under 10 microns;
(4) Tabulation of response data
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153
'(6) The LCSO or LDSO of the microhial agent (calculated for
an exposure of on* hour or from lethality data) for each sax and
each test substance;
(?) Specification of th« method usad for LCSO or LD50
calculation;
(8) Tha 95 parcant confidanca intarval for tha LCSO or LD50>
(9) Tha dose-response curva and alopa (with confidanca limits);
(10) Tha finding* from a histopathological study, if conducted,
including a complete record of lesions and abnormalities observed,
and tha hiatological characterisation of each kind of lesion or
abnormality obeerved, naming those which apparently caused death or
moribund!ty;
(11) The gross pathological findings; and
(12) Evidence of microorganism dissemination, replication,
and survival in »«<•»•» tissues, organs, and fluids, particularly in
nasal passages, tracheae, bronchi, and lungs.
(13) Results of the assay for specific antibody production
shall be reported in tests exceeding 14 days duration.
(d) Tier progression.
(1) Ho further testing involving the respiratory route of
exposure using bacteria or fungi is necessary.
(2) If evidence of infactivity, organism persistence or
replication, or toxicity is observed following the inhalation
infectivity/toxicity study using viruses or protozoa is found,
then a Tier II acute inhalation study (§ 152-41) with the micro-
bial agent shall be required by 40 CFR § 156.165. Tier II testa on
protozoa will use a different species than that used in Tier I and
tests on viruses will use newly weaned mice or newly weaned hamsters.
In addition, the teratogenieity study (§ 152-47) shall be required
for viruses as specified in 40 CTR $ 158.165.
(3) If no evidence of acute inhalation infectivity, persistence,
replication, or toxicity is observed, then additional testing is
not necessary.
(a) References. The following texts and articles give general
information as well as sufficient detail to develop and carry our
inhalation toxicity/infactivity studies.
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154
(1) General references.
(i) .Altaian, P.I.., «t al. 1958. Handbook of Respiration.
Aero Medical Laboratory. Wright-Patterson Air Fore* Baa*,
Ohio. (Includes extensive data on human* and aniaala.)
(ii) Casarett, L.J., and J. Doull. 1975. Toxicology: Basic
Science of Poisons. MacMillan Publishing Co., Inc. New York.
(Good text on general toxicology, includes Chapter 9, "Toxicology
of the Respiratory System".)
(iii) Comroe, J.H. 1974. Physiology and Respiration. 2nd
Ed. Year Book Medical Pub. Chicago. (Includes the anatomy,
physiology, physiological testing, and pathology of the lungs in
humans.)
(iv) Hatch, T.G., and P. Gross. 1964. Pulmonary Deposition
and Retention of Inhaled Aerosol* Academic Press* New York.
(Comprehensive text which deals with the anatomy, physiology,
deposition and retention, and pathological changes in the lung.)
(v) ICFP Committee, P. Morrow (Chairman). 1966 and 1967.
Deposition and retention mo'dels for internal dosimetry of the
human respiratory tract. Health Phys. 12:173-207 (1966). Errata
and revisions to report, Health Phys. 13:1251. (1967).
(vi) Ignoffo, C.M. 1973. Effects of entomopathogens on
vertebrates. Ann. N.Y. Acad. Sci* 217:141-172.
(2) Advanced Monographs.
(i) Mercer, T.T., P.I. Morrow, and W. Stober. 1972. Assess-
ment of Airborne Particles. Charles C. Thomas. Springfield, 111.
(ii) Walton, W.H., ed. 1970. Inhaled Particles III. Proceed-
ings of an International Symposium organized by the British Occupa-
tional Hygiene Society. London. September 14-23, 1979. Volumes
I and II. Unwin Brothers Limited. Gresham Press. Surrey, England.
(iii) Walton, W.H., ed. 1975. Inhaled Particles. IV. Pro-
ceedings of an International Symposium organized by the British
Occupational Hygiene Society. London. September 22-26, 1975.
Volumes I and II. Pergamon Press, Mew Yorh.
(3) Exposure Systems.
(i) Drew, R.T., and S. Laskin. 1973. Environmental Inhala-
tion Chambers. Pp. 1-41 in Methods of Animal-Experimentation, Vol. IV.
Academic Press Inc., New York and London.
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155
(ii) Praser, D.A., R.E. tales, M. Lippmann, and H.E. Stokinger.
1959. Exposure Chambers for Research in Aniaal Inhalation. Public
Health Service Monograph No. 57. U.S. Government Printing Office.
Washington* O.C.
(iii) Sinners, R.G., J.K. Burkaxt, and C.L. Paste. 1968.
Aninal Inhalation exposure chambers. Arch. Environ. Health 16t194-206.
(iv) Phalen, R.F. 1976. Inhalation exposure of animals.
Znviron. Health Perspeet. 16i17-24.
(v) Roe, P.J.C. 1958. Inhalation tests; in Modern Trends in
Toxicollogy, Vol. 1. Borland, X. and R. Goulding, eds. Appleton-
Century-Crofts, New York.
(4) Generating1 Systems. Gas and vapor generation is relatively
simple compared to the large number of different systems needed to
generate aerosols of solids and liquids. Besides the following
references, all of the advanced monographs in paragraph (3) contain
descriptions of generating systems.
(i) Drew, R.T., and M. Lippman. 1972. Section X. Calibra-
tion of Air Sampling Instruments. II. Production of Test Atmospheres
for Instrument Calibration; in Air Sampling Instruments. 4th
Ed. Am. Conf. Gov. Ind. Bygienists.
(ii) Praser, D.A., et. al. 19S9. Exposure Chambers for
Research in Animal Inhalation. Public Health Monograph No. 57.
Supt.Doc., U.S. Gov. Print. Off. Washington, D.C.
(iii) Raabe, O.G. 1970. Generation and Characterization of
Aerosols. Pg. 123 in Inhalation Carcinogenesis. OSAZK Conf-691001.
M.G. Hanna et al. Clearinghouse for Federal Scientific and Tech-
nical Information. Springfield, Va.
(5) Sampling Methods.
(i) Ldppman, M. 1972. Respiratory Dust Sampling. Section G
in Air Sampling Instruments. 4th Ed. American Conference of
Governmental Hygienists, Cincinnati.
(ii) Morrow, P.E. 1964. Evaluation of inhalation hazard
based upon the respirable dust concept and the philosophy and
application of selective sampling. Amer. Ind. Hyg. Assoe. 25:213-
236.
(iii) National Institute of Occupational Health and Safety.
1973. The Industrial Environment. - Its Evaluation and Control.
Supt.Doc., Gov. Print. Off., Washington, D.C. (Has several good
chapters on sampling, and on chemical, instrumental, and physical
analyses of atnospheres.)
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156
(iv) Peterson, C.M. 1972. Aerosol Sampling for Particle
Size Analyses* Section T - Respiratory dost sampling in Air Sampl-
ing Instruments* 4th Ed. American Conference of Governmental
Rygieaists, Cincinnati*
(v) Preining, O., D. Sheesley, N. Djordevic, et al. 1967.
The size distribution of aerosols produced by air blast nebuliza-
tion. J. Colloid and Interface Sci. 23s 3.
(vi) Silvarman, L., C.E. Billings, M.W. First, et .al. 1971.
Particle Sice Analysis in Industrial Hygiene. Academic Press,
Inc., Hew York and London* (Sampling size analysis and instrumen-
tation with an emphasis on hygiene and air cleaning.)
(6) Pulmonary function testing. Pulmonary function tests have
been widely used in the evaluation of human respiratory function
but less widely used in animal research. Such analyses may be
required to indicate subtle damage to the pulmonary system.
(i) Alarie, T*, A. Krunm, H. Jennings, R. Haddock, et al.
1971. Distribution of ventilation in cyanomologus monkeys* Arch.
Environ* Health 22:633. (Illustrates testing in a primate.)
(ii) Amdur, M.O., and J. Head. 1956. Mechanics of respira-
tion in unanesthetized guinea pigs. Am. J. Physiel. 192:364.
(Illustrates function testing in a rodent.)
(ill) Comroe, J.H., et al. 1962. The Long, Clinical Physiology
and Pulmonary Function Tests. 2d Ed. Tear Book Msd. Publ.,
Inc., Chicago. (Provides a discussion of pulmonary testing along
with anatomy and physiology of the human respiratory system.)
(iv) Comroe, J.H. 1965. Physiology of Respiration. JTear
Book tied. Publ., Inc., Chicago. [Similiar to Comroe et al., 1962
(above).}
(v) Mauderly, J., and J. Pickrell. 1973. Pulmonary Function
Testing of Onanaesthetized Beagle Dogs; in Research Animals in
Medicine. L. Harmison, ed. DBZH Pub. No. HZH 72-333. (Illustrates
function testing in the dog.)
§ 152-33 Intravenous, intraeerebral, and intraperitoneal toxicity/
infactivity studies with microbial pest control
agents; Tier I.
(a) Whan required. Data from the following tests are- required
by 40 C?R 158.165 to support the registration of. each manufactoring-
use product and each end-use microbial pest control agent as follows:
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157
(1} Intravenous ("IV"} infactivity study for bacterial and
viral agents;
(2) Intracerebral ("1C") infactivity study for viral and
protozoan agents; and
(3) Intraperitoneal ("IP") infactivity study for fungal and
protozoan agents.
(b) Test standards. In addition to the general standards set
forth in $ 150-3 of this subdivision and § 80-3 of Subdivision F,
studies outlined in this section should meet the following standards:
(1) Substance to be tested. The technical grade of each
active ingredient [termed purest infective form ("PIT") for viruses]
shall be tested.
<2) Species. Testing for studies required by this section
should be performed with the following test animals:
(i) Newly weaned mouse and newly weaned hamster for IV study
with bacteria and viruses;
(ii) Mouse and one other species for IF study with fungi;
{iii) Newborn mouse and newborn hamster for 1C study with
viruses; and (iv) Mouse and rabbit for 1C and IP studies with
protozoa.
(3) Sex. Approximately equal numbers of males and females
should be used.
(4) Inmunodepression. One half of the animals used in these
studies should be iominodepressed.
(5) Number of animals. An adequate number of animals should
be injected with a single high dose of test substance to permit
periodic sacrifice at 1, 2, 3, and 4 weeks (termination of study)
post-treatment. At least 5 animals per sex/ species/ and state of
iauunodepression per sacrifice for the hamsters and mice should be
used.
(6) Control animals. A control group of animals (one half
ianinodepressed) should be injected with the test-vehicle containing
killed test organism. All control animal* should be sacrificed at
four weeks post-treatment. At least five animals per sex, species,
and state of immunodepression should be used*
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158
{7) Conduct of test.
(i) Application. Approximately 0.05 ml (for mice and for
newborn and newly weaned hamsters) or 0.1 ml (for other animals)
of test substance should be injected in accordance with standard
procedures and sites for XV, 1C, aad IP tests. The test substance
should be injected in undiluted form. If the test substance is a
solid, it should be dissolved in a alnlasl amount of physiological
saline.
(il) Duration of observation* Surviving animals should be
observed for at least four weeks after dosing, or until all signs
of reversible infactivity in survivors subside, whichever occurs
later.
(iii) Observations. The animals should be observed frequently
during the day of dosing and checked at least once each morning
and late afternoon thereafter* The following should be observed
even if the animals recover completely from the exposure: nature
and onset of all gross or visible clinical signs of illness such
as unkempt appearance, altered feeding habits/ weight loss, physical
depression, depression, and similar expressions.
(iv) Assay for production of specific antibodies. An assay for
the production of specific antibodies should be performed in each
study, excepting those using newborn or newly weaned test animals.
(v) Sacrifice and necropsy. All test animals surviving at the
end of the observation period should be sacrificed. All test animals,
whether dying during the test or sacrificed, should be subjected to
a complete necropsy. In addition to reporting gross pathology,
evidence of organism survival and/or multiplication in blood and
at distant sites such as the spleen, liver, lung, and brain should
be determined by eulturing tissues on laboratory or other suitable
media or host to provide qualitative and quantitative evidence
of possible survival and multiplication of the test organism(s).
(6) Dose quantification. liters of microbial preparations ad-
ministered to test animals should be determined by plating dilutions
on suitable media or host organisms for enumeration of viable
organisms.
(c) Data reporting and evaluation. In addition to the infor-
mation required by $ 80-4 of Subdivision F, the test report should
include the following informations
(1) Tabulation of response data by species, sex, and state of
immunodepression as to numbers of animals exhibiting signs of
infeetivity and/or confirmed recovery of test animal per number of
animals exposed;
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159
(2) The time of death after doling;
(3) Results of gross pathological examinations, including
recovery adn approximate numbers of viable test microorganisms
found in various cultured tissues; and
(4) Identification of the test microorganism, including:
(i) Genusr species, serotype and strain (to the extent
possible) according to current acceptable taxonomy; and
(ii) The percent of unknown fermentation solids or other
materials present to account for 100 percent of the sample tested.
(5) Results of the assay for production of specific antibodies
when applicable.
(d) Tier progression*
(1) If evidence of prolonged survival or replication in mam-
malian hosts and significant damage to mammalian cells is observed
when viruse* are tested, then data on teratogenicity testing
(§ 152-47) are required by 40 CFR 158.165.
{2} If evidence of infectivity or organism persistence,
replication, or toxicity is observed when protozoa are tested, then
additional intraperitoneal and intracerebral testing (§ 152-43} and
acute oral testing (§ 152-40) is required by 40 CFR 158.165.
(3) If evidence of infectivity is observed (e.g., prolonged
survival and/or replication) when bacteria or fungi are tested,
then the virulence enhancement test in Tier II (§ 152-48) using the
mouse or hamster shall be required by 40 CFR 158.165.
(4) If no evidence of infectivity or prolonged survival is
observed, -hen further testing is not necessary.
(e) References. The following references contain useful infor-
mation for developing an acceptable protocol.
(1) Hansen, G.O. 1973. Elimination rate of Bacillus
thuringiensis spores administered to mice. Abbott Laboratories
T-9, Project no. 70111. Information supplied to the subcommittee
by Abbott Laboratories, North Chicago, 111.
(2) Ignoffo, C.M. 1973. Effects of entomopathogens on
vertebrates. Ann. N.Y. Aead. Sei. 217:141-164.
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160
(3) Lasutnna, C., and L. Jones. 1963. Lethality for mice of
vegetative and spar* form* of Bacillus c«r«u« and Bacillus cereus-
like insect pathogens injected intraperitoneally and subeutaneously.
J. Bacteriol* 85: 532-535.
(4} Leise, J.M., C.H. Carter, H. Friedlander, and S.W. Freed.
1959. Criteria for the identification of Bacillus anthraeis.
J. Bact«riol.77t 655-660.
§ 152-34 Prinary dermal Irritation study with microbial pest
control agents; Ti«r I.
(a) Whan required. Data on primary dermal irritation are
required by 40 CPR § 158.165 to support the registration of each
manufacturing-use product and each end-use product.
(b) Test standards. The general standards sat forth in | 150-3
of this subdivision and $ 80-3 of Subdivision P should apply. In
addition to these general test standards/ a primary dermal irritation
study should meet the following standards;
(1) Substances to be tested.
(i) The manufacturing-use product shall be tested to support
rh« registration of a manufacturinguse product.
(ii) The end-us* product shall be tested to support the regi-
stration of an end-use product.
(2) Species and age. Testing should be performed with young
adult guinea pigs or rabbits.
(3) Condition of test substances.
(i) If the substance is a liquid, it should be applied undiluted.
(ii) If the test substance is a solid, it should be slightly
moistened with physiological saline before application.
(4) Number of animals. At least six animals shall be used.
(5) Kumber and selection of dose levels. A dose of 0*5 ml of
liquid or 0.5 g of solid or semi-solid microbial preparation is to
be applied to each application site.
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161
(6) Dose quantification. Titers of microbial suspension admin-
istered to test animals should be performed by plating dilutions on
laboratory surface media or other suitable media or host organism
for enumeration of viable organisms.
(7) Control groups.
(i) A vehicle control group is recommended if the vehicle
is known to cause any toxic dermal reactions or if there is insuf-
ficient information concerning the dermal effects of the vehicle.
(ii) Separate animals are not necessary for an untreated
control group. lach *"t»Tl serves as its own control.
(8) Conduct of test. The test substance is introduced
under one-inch square gauxe patches. The patches should be applied
to two intact and two abraded skin sites on each animal. In all
animals, the application sites should be clipped free of hair. The
abrasion should penetrate the stratum corneum, but not the dermis.
A wrapping material such as gauze covered by an impervious, non-
reactive rubberized or plastic material should be used to retard
evaporation and to keep the test substance in contact with the
skin. The animals should be restrained. The test substance must be
kept in contact with the skin for 24 hours. At the end of the
exposure period, the wrapping should be removed and the skin wiped
(but not washed) to remove any test substance still remaining. It
may be necessary to rinse off the material if colored test substances
are used.
(9) Observation and scoring. Animals shall be observed and
signs of erythema and edema shall be scored at 24 hours and 72
hours after application of the test substance. The irritation is
to be scored according to the technique of J.H. Draize (1959).
Observation for irritation and scoring of any irritation shall
continue daily until all irritation subsides or is obviously
irreversible.
(c) Data reporting and evaluation. In addition to the
applicable general information required by $ 80-4 of Subdivision
F [excepting paragraphs (b)(2)(ill)(A) and (b)(2)(vii)], the test
report shall include the following informationi
(1) In tabular form, the following data for each individual
animal and averages and ranges for each test groupt
(i) Scores for erythema and edema at 24 hours, at 72 hours,
and at any subsequent observation, andi
(ii) Primary skin irritation scores according to the technique
of Draize.
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162
(d) Tier progression.
(1) Mo further testing is necessary for manufacturing-use
products.
(2) Zf evidence of primary dermal irritation is observed (marked
edema or broad erythema) in tests conducted on the end-use formulated
product, then:
(i) Primary dermal irritation studies in the guinea pig shall
be required by 40 CFR 158.165 using use dilutions of the end-use
product {{ 152-44).
(3) Zf no evidence of primary dermal irritation is observed,
then further testing is not necessary.
§ 152-3 5 Primary eye irritation study vith microbial pest control
agentst Tier I.
(a) When required. Data on primary eye irritation are required
by 40 CFR 158*165 to support the registration of each manufacturing-
use product and each end-use product*
(b) Test standards. The general standards set forth in $ 80-3
of Subdivision F apply. In addition to these general test standards,
a primary eye irrigation study should meet the following standards:
( 1 ) Substances to be tested.
ti) The manufacturing-use product shall be tested to support
the registration of a manuf acturinguse product.
(ii) The end-use product shall be tested to support the
registration of an end-use product.
(2) Species and age. Testing should be performed using young
adult rabbits.
(3) Condition of test substance.
(i) Zf the test substance is in liquid form, it should be
applied undiluted.
H the test substance is a solid, it should be slightly
moistened with physiological saline before application.
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163
(4) Numbers of animals. At least nin« animals should be used.
(S) Number and selection cf dose. A dose of 0.1 ml of liquid
or 100 ng of solid should normally be applied to each teat eye*
smaller quantities may be used when the standard quantities would
be lethal, or when 100 mg of the solid cannot feasibly be admini-
stered to the eye.
(6) Dose quantification* Titers of microbial suspensions to
test animals should be perforated by plating dilutions on laboratory
surface or other suitable media or a host organism for enumeration
of viable organisms.
(7) Caging* Caging should be designed to minimize exposure to
sawdust, wood chips, and other extraneous materials that might
enter the eye*
(8) Conduct of test. The test substance should be placed on
the everted lower lid of one eye; the upper and lower lids are then
then to be gently held together for 1 second before releasing to
prevent loss of material. The other eye/ remaining untreated,
serves as a control. Die treated eyes of 6 rabbits shall remain
unwashed. The remaining 3 rabbits shall receive test material, and
then the treated eye should be flushed for one minute with lukewarm
water starting no sooner than 20-30 seconds after instillation. A
local anaesthetic to reduce pain in test animals may be used prior
to administration of the test substance, provided that evidence can
be presented indicating no significant difference in toxic reaction
to the test substance will result from use of the anaesthetic.
(9) Observation and scoring.
(i) Observation. Headings of ocular lesions should be made at
24, 48, and 72 hours after treatment and at 4 and 7 days after
treataent. Readings should be made every 3 days thereafter, if
injury persists, for at least 21 days after treatment or until all
signs of reversible toxicity subside. Grading and scoring of
irritation are to be performed in accordance with Table 5 [from
Draize et al. (1965)]. The most serious effects, such as pannus or
blistering of the conjunctivae and other effect* indicative of
corrosive action should be reported separately.
(c) Data reporting and evaluation. In addition to the
applicable general information required by J 80-4 of Subdivision F, the
test report should include, In tabular form, the following data for
each individual animal and the averages and range for each test
group {eyes washed and unwashed):
(1) The primary eye irritation score at 24, 48, and 72 hours
and 4, 7, and 21 days and any other readings; and
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164
A description of any serious lesions*
(d) Tier progression.
(1) No further testing is necessary for manufacturing-use
products*
(2) Zf evidence of primary ocular irritation (e.g., severe
ocular lesions) is observed in tests conducted on the end-use
formulated product.', then primary ocular irritation studies in the
rabbit (§ 155-45) shall be required by 40 CTR 156.165 using use-
dilutions of the end-use formulated product.
(3) Zf no evidence of primary ocular irritation is observed,
then further testing is not necessary.
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165
5—WEIGHTED SCORES FOR GRADING THE SEVERITY OF OCDIAR LESIONS
I. Com**
(A) Opacity - degree of density (area taken for reading)
- scattered or diffuse area, detail* of iri« clearly visible. ..... 1
- Easily discernible translucent areas, details of iris slightly
obscured ................. 2
- Opalescent areas, no details of iris visible, sise of pupil barely
discernible ...« ...... .....3
- Opaque, iris invisible 4
(B) Area of cornea involved
- One quarter (or less) bat not sero 1
• Greater than one quarter nut 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 maximum - 80
II. Iris
(A) Values
• Folds above normal, congestion, swelling, circuncorneal injection (any
one or all of thes* or combination of any thereof), iris still reacting
to light {sluggish reaction is positive) ....1
- No reaction to light/ hemorrhage; gross destruction (any one or all of
these) 2
Score equals A x 5 Total possible maximal • la
III. Conjunctiva*
(A) Redness (refers to palpebral conjunctivas only)
- Vessels definitely injected above normal 1
- More diffuse, deeper crimson red/ individual vessels not easily
discernible 2
- Diffuse beefy red 3
(B) Chemosis
- Any swelling greater than normal (includes nictitation membrane) ... 1
- Obvious swelling with partial eversion of the lids 2
- Swelling with lids about half closed 3
- Swelling with lids about half closed to completely closed 4
(C) Discharge
- Any amount different from normal (does not include small amount
observed in inner canthus of normal animals) •_»•• 1
- Discharge with moistening of the lids and hairs just adjacent to the
lids 2
- Discharge with moistening of the lids and considerable area around the
•ye . 3
Score equals (A •*• B + C) x 2 Total possible maximum » 20
The maximum total score is the sun of all scores obtained for the cornea,
iris/ and conjunctivae.
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152-36 Hypersensitivity study with microbial pest control agents;
Ti«r I.
(a) tlhen required. Data from a hypersensitivity study are
required by 40 CTR $ 158.165 to support the registration of each
end-use product for which commonly recognized use practices will
result in repeated human contact by inhalation or dermal routes
and each manufacturing-use product which legally may be used to
formulate such an end-use product.
(b) Test standards. In addition to the applicable general
standards set forth in | 150-3 of this subdivision and § 80-3 of
Subdivision F, a hypersensitivity study shall meet the following
standards.
(1} Substance to be tested.
(i) The manufacturing-use product shall be tested to support
the registration of a manufacturing-use product.
(ii) The end-use product shall be tested to support the
registration of an end-use product.
(2) Condition of test substance. The test substance should be
applied undiluted. If the test substance causes marked irritation,
it should be diluted with physiological saline until a concentration
is found which produces only slight irritation. If the test
substance is a solid to be injected intradermally, it should be
dissolved in a minimum amount of physiological saline*
(3) Species. The test should be performed in the hamster or
guinea pig.
(4) Age and sex. Young adult males should be used when albino
guinea pigs are tested. Young adults of either sex may be used
when hamsters are tested.
(5) Number of animals. At least 10 animals should be used.
(6) Pose quantification. Titers of microbiml^^uspensions
should be determined by plating dilutions on laboratory surface or
other suitable media or a host organism for enumeration of viable
organisms.
(7) Number and selection of dose levels, (i) An initial dose of
0.05 ml should be injected intradermally. This dose shall be followed by
injection of 0.1 ml three times weekly on alternate days for three weeks,
so that a total of ten treatments is administered. Two weeks after the
tenth sensitizing treatment, the animals should be challenged by a final
injection (L*ndsteiner and Jacobs/ 1935);
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(8) Control*, (i) A positive control, using * kno%m
sensitizing agent, is recommended; «nd
(ii) A concurrent vehicle control group is not required.
(9) Conduct of test.
(1} Preparation of test animals. Hair is removed first
by clipping and then by shaving to form a strip running from flank
to trunk along each side of each animal. This procedure should be
repeated as necessary.
(ii) Intradermal injection. After preparation of the test
animal, the test substance is injected intradermally. The
first sensitizing injection is be made at one end of one strip.
The succeeding injections should be made by moving along the shaved
strip choosing a new location for each treatment.
(10) Observation and scoring. Erythema, edema, and other
lesions are scored at 24 hours and 48 hours following each
application, according to the standard method (Draize, 1959).
(c) Data reporting and evaluation. In addition to the
applicable basic information in $ 80-4 of Subpart F, the
following information should be reportedi
(1) Tabulated scores for each animal for erythema and edema
at 24 and 48 hours post-application or post-injection; and
(2) Tabulated average scores from all sensitizing treatments,
and the score of the challenge treatment.
(d) Tier progression. No tier progression from the
hypersensitivity study is necessary.
§ 152-37 Hypersensitivity incidents with microbial pest control
agents* Tier I.
(a) When required. Data on incidents of hypersensitivity 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 with the toxicology data supplied in support of an appli-
cation for registration. Tor reporting of incidents talcing place
after registration, refer to the requirements in.connection-with.
sec. 6(4)(2) of FX7KA.
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(b) Reporting. The reporting requirements for these inci-
dents should be the same MI those for conventional chemical pesti-
cide incident reports as specified in the Pesticide Incident Report
form (EPA form number 8550-5, 0KB form number 158-R00081. the
following information should be provided if available:
(1) The name of the microbial agent;
(2) The length of exposure to the agent;
(3) The time, date and location of exposure to the agent;
(4) The situation or circumstances under which exposure to
the agent occurred; and
(5) Any clinical observations.
§ 152-38 effects of microbial pest control agents on the cellular
iaaune response system: Tier I.
(a) Test description. The following tests of effects on
ioBunocompetence are included in the cellular inune response
studies with microbial pest control agents:
(1) Blood cell count:
(2} Leukocyte response (B and T cells};
(3] Total leukocyte number (T and B cell ratios);
(4) Macrophage number and function; and
(5) Serum protein determination.
(b) When required. Data on cellular immune response studies
are required by 40 QTR J 158*165 to support the registration of each
end-use product and each manufacturing use product. See 40 C7R
§ 158.50 and | 158.165 to determine whether these data must be sub-
mitted; Section ZZ-B of this subdivision contains an additional
discussion of the femulators' exemption and who* as a f-eneral
rule, is responsible for submission of the requir«4-4*ta.
(e) Test standards* In addition to the test standards set
forth in | 150-3 of this subdivision, the following standards should
be met:
(1) Test substance. The technical grade of the microbial
agent shall be tested.
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169
(2) Dosage. At least three doses of * given microbial agent
should be separately administered to test mice prior to performing
the cellular immune response evaluations.
(3) Test methods. The test aethods for the five different
cellular Untune response tests described in (i) through (v) apply
for any candidate microbial agent (i.e./ bacterial, fungi/ virus/
or protozoa):
(i) Blood cell count, three groups of 10 »ale and 10 fesvale
mice each are separately injected (intraperitoneally) with a 0.5 ml
dose containing different concentrations of test substance. One
group of nice are injected with undiluted test substance, one with
test substance diluted lOx, and a third group injected with agent
diluted 100x. Three groups of five alee each injected with
physiological saline should serve as controls. Routine blood
counts should be performed at 15 and 30 days after exposure.
Standard hemacytometer assays should be utilized to determine the
total number of peripheral blood leukocytes as well as differential
counts.
(ii) Leukocyte response (T and B cells). Three groups of
treated mice and three groups of control mice are prepared as
described above (i) for blood cell counts. Fifteen and 30 days
after treatment, treated and control nice are to be tested for
absolute peripheral blood T and B lymphocyte numbers, and/or their
ratio, in order to ascertain whether or not there has been a
significant shift in the populations of these cells. An iamunc—
fluorescent antibody test should be used to determine the number
of peripheral blood leukocytes exhibiting abnormal numbers of
total surface imaunoglobulins. The E-rosette assay with sheep
erythrocytes should be used as a standard technique for ascer-
ta.uia.ng the number of T cells in the peripheral blood of mice.
(iii) Total leuxpeyte number (T and B cell ratios). Three
groups of treated alee and three groups of control alee are pre-
pared as described above (i) for blood cell counts. At several
time intervals after treataent, such as IS and 30 days/ treated
and control alee shall be tested for absolute peripheral blood B
and T lymphocyte number and/or their ratio in order to ascertain
whether there has b«en a significant shift in the population of
these cells. An inmunofluorescent antibody test vising antimouse
immunoglobulin serum is a standard method and wvif^Jst* used to
determine the number of peripheral blood leukocytes which exhibit
changes In surface immunoglobulins characteristic for B lymphocytes.
For the T cells, indirect iamunofluorescence assays using standard
techniques should also be used, as in $ 152-18(c)(4)(ii) and
(iii). (S«e references for cellular immune response test
§ 152-18.)
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170
(iv) Maerephage number and function* On* group of at least S
mice are each injected intraperitoneally with 0.5 ml of undiluted
test substance. Fifteen and 30 days after exposure, the number and
percent of peritoneal and splenic macrophages should be determined
by standard phagocytic index tests, such as the uptake of latex
diffusion assays and electrophoresis. Amounts of individual classes
of immunoglobulins nay be determined by inmunoelectrophoresis to
determine levels of Zg G, M, A, and D.
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171
testing in the model systems, * minima -of five plaque-forming
unit* per cell is required when a plaque assay for the virus la
available, or seven LDSO's per cell when a plaque assay for the
virus ia not Available. If not feasible, the reasons for lack of
compliance should be noted and submitted, and maximal amounts of
infectious virus compatible with tissue culture systems shall be
assayed.
(2) Cell cultures. The following cells should.be used: 1
human line, e.g., WI38; ^ human primary cell type, e.g., foreskin?
1 human continuous line, e.g., lelAr 1 priaata continuous line,
e.g., monkey C7-1 or BSC-1 and, for assessment of possible slowly
developing virus-cell interactions that could lead to virus
persistence and/or malignant transformation, mouse 3T3 cells.
(3) Number of test systems. Replicate cultures containing
a minimise of 10s cells each should be inoculated in each test.
(4} Observation of gross morphological changes (cytopathie
effects). Two confluent plates should be examined dally by light
microscopy for a minimum of 14 days for human and primate lines,
and weekly intervals for up to one month for the 3T3 cells.
(5) Inhibition of cell division. Two sub-confluent plates
should be used for measuring the ability of infected cells to grow
to confluency after inoculation.
(6) Bioassay of culture fluid. Cells and culture fluid from
replicate confluent plates should be assayed for infectious virus on
alternate days for 14 days after inoculation. The bioassay chosen
should be the most sensitive available for detecting infectious
virus.
(7) Assay for decay of input virus and potential appearance
of viral proteins and nucleic acid, (i) A sensitive quantitative
issmunological test (e.g., the enzyme-linked iamunosorbent assay)
should be performed on replicate cultures on alternate days for 14
days.
(ii) A sensitive quantitative molecular hybridization test
for viral nucleic acid (e.g., kinetics of reassociation of highly
labeled viral DNA probe using infected cell UNA as driver) on
replicate cultures on alternate days for 14 days should be performed.
(8) Controls. In each instance, mock-infected cultures should
be similarly analyzed. For each series of tests the inoculum will
be tested in the permissive cell line or insect host as a positive
control and for direct reference to the data obtained from the
vertebrate cell lines.
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172
(c) Data reporting and evaluation. The following information
should be provided on each test:
(1) Cytopathic effects (CPE) in the cell monolayers.
(i) The appearance of CPE in the cell monolayers described to
differentiate between cell destruction induced by the test virus
and nonspecific effects.
(ii) Results of tube culture inspection for microscopic
evidence of cytopathic effects recorded as:
1+ • suggestive of virus-induced morphologic changes;
2+ • definite morphologic changes i
3* • more than 50% cell degeneration; or
4+ • complete cell destruction.
(iii) The TCII>50 value calculated by a statistical method
(i.e., the Reed and Meunch Method). For computation of the
infeetivity results, only tubes showing a 2+ CPE or greater are
considered to be infected.
(2) Inhibition of cell division.
(i) The procedure used for the study of cell-division inhibition
should be detailed.
(ii) The initial cell-number seeded for adequate proliferation
determined by either henocytometar counting or electronic enumeration.
(iii) Results of the cell-number of infected and control cul-
tures monitored during the of study, expressed as a percentage as
follows:
Average cell count, infected cultures - initial cell count j 100
Average cell count, control cultures - initial cell count
(iv) The percentage of confluency of cell monolayer* from
infected and control cultures (determined after an appropriate
period of incubation).
(v) Any evidence of mitotic process prevention or interference
with chromosomal replication.
(3) Bioassay of culture fluid.
(i) Susceptible host sytem used for viral detection.
(ii) Records of the dilutions in the virus assay (e.g., 10~1,
10~2). The serial dilution method is required.
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173
(liU The percentage infectivity calculated from the number of
infected host cells following inoculation with an appro-
priate series of consecutive virus dilution.
(4) Data from aa assay for decay of input virus.
(i) Identity of viral antigens that do not grow wall in call
culture*
(A) Detail* of procedure* of the Enzyme-Linked Immunosorbent
Assay (rLISA) for viral detection.
(ii) Report the cellular location of viral antigens in the
infected cell culture.
(A) Details of procedures of the iamunofluorescence technique.
(5) Assay for potential appearance of viral proteins and nucleic
acid.
(i) Details of procedures of the DNA-DNA reassociation kinetics.
(d) Tier progression.
(1) If tissue culture tests show evidence of replication or
persistence of the viral agent, or damage to host cells, then
additional testing in Tier II is required by 40 C?R 158.165.
The specific Tier II test requirements will depend on the results
of the tissue culture test and will be determined after consultation
with the Agency.
(2) If tissue culture tests show no evidence of replication
or persistence of the viral ager.t/ or damage to host cells, then no
further testing is necessary.
(e) References.
(1) Bullock, S.L., and r.w. Walls. 1977. evaluation of some
of the parameters of the enzyme-linked isonanospecific assay. J.
Infect. Pis. 136: S279-S285.
(2) Engvall, E., and P. Perlaann. 1972. Enzyme-linked
aomunosorbent assay. CLXSA. III. Quantification of specific
antibodies by enryme-linked antlglobulin in antigen-coated tubes.
J. lamunol. 109:129-135.
(3) Gelb, L.D., D.E. Kohne, and M.A. Martin. 1971. Quantita-
tion of simian virus 40 sequences in African Careen monkey, mouse
and virustransformed cell genomes. J. Mol. Biol. 57:129-145.
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174
(4) Goldman, M. 1968. Fluorescent Antibody Method*. Academic
Press, Inc. New York, N.Y.
(5) Hermann, J.E., R.M. Hendry, and M.F. Collins. 1979.
Factors involved in enzyme-linked inmunoassay of viruses and
evaluation of the method for enteroviruses. J. Clin. Mierobiol.
10:210-217.
(6) Rigby, P.W.J., M. Kieckmann, C. Rhodes, and P. Berg.
1977. Labeling deoxyribonucleie acid to high specific activity in
vitro by nick translation with DMA polymerase I. J. Mol. Biol.
113:237-251.
(7) Hold, W.S.M., M. Green, and J.K. Mackey. 1978. Methods
and rationale for analysis of hiaan tisnors for nucleic acid sequences
of oncogenic human DMA viruses. Methods Oncer Res. 15:69-161.
(8) Yolken, R.H., H.W. Kim, T. Clem, R.6. Hyatt, A.R. Kaliea,
R.M. Chanock, and A. 2. Kapikian. 1977. Bazyme-linked inmunosorbent
assay (ELISA) for detection of human reovirus-like agent of infantile
gastroenteritis. Lancet 2:263-266.
Group B-2: Tier II Testing.
§ 152-40 Acute oral toxicity/infeetivity studies with viral or pro-
tozoan agents; Tier II.
(a) When required.
(1} End-use formulated products. Data on the acute oral
infactivity of viral or protozoan agents (Tier II) are required
by 40 CFR § 158.165 to support the registration of each end-use pro-
duct for which survival, replication, infectivity, toxicity, or
persistence of the microbial agent (virus or protozoa) is observed
in the test animals treated in the Tier I acute oral infectivity
tests ($ 152-30) or the intraperitoneal/intracerebral injection
test for protozoa (f 152-33).
(2) Manufaeturinyuse products. Data on the acute oral infec-
tivity of viral or protozoan agents (Tier XX) are-revtffifared by 40 CFR
§ 158.165 to support the registration of each manufacturing-use product
for which survival, replication, infectivity, toxicity, or persistence
of the microbial agent (virus or protozoa) is observed in the test
animals treated in the comparable Tier X acute oral infectivity tests
(i 152-30) or the intraperitoneal/intracerebral injection test for
protozoa ($ 152-33).
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(to) Test standards, in Addition to the applicable general stan-
dard* set forth in $ 150-3 of thi* subdivision and $ 80-3 of Subdivi-
sion F, tha acute oral infactivity test with protozoa or viruses should
be conducted in accordance with the standards set forth in § 153-30
of this subdivision/ with the following exceptionst
(1) Substance to be tested. (i) The end-use product shall be
tested to support the registration of an end-use product*
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176
of -all -gross or visible clinical signs of illness such as elevated
temperature, unkempt appearance, altarad feeding habits, weight
loss, and various forms of distress and physical depression.
(iii) Sacrifice and necropsy. All test animals surviving at ter-
mination of the observation period should be sacrificed. All test
animals, whether dying during the test or sacrificed, should be sub-
jected to a complete gross necropsy. If pathology is observed,
organs and tissues must be examined microscopically. In addition to
gross pathology/ the following should be determined» microorganism
dissemination, replication and survival in animal tissues, organs,
and fluids, including survival in the intestinal tract. Samples
should be cultured on laboratory surface or other* suitable media or
a host organism to provide qualitative and quantitative measurements
of survival and multiplication of the microorganism. In addition, for
tests of viral agents/ samples of blood, spleen, liver, heart, skeletal
muscle, lung, and small intestine should be examined for infactivity.
These samples should also be assayed by sensitive molecular techniques,
to be performed using radioiamunoassay, enzyme-linked lamunosorbant
assay, or nucleic acid hybridization tests*
(c) Data reporting and evaluation* In addition to the informa-
tion required by § 80-4 of Subdivision 7 and $ 152-30 of this subdivi-
sion/ the test report should include the following information:
(1) When using the serologic and nucleic acid tests to detect
viruses, the sensitivity, specificity, and limits of each assay
shall be defined by a comparison to a standard assay using the permis-
sive system.
(d) .Tier progression. (1) A chronic feeding study (§ 152-50)
is required by 40 OPR $ 158.165 for viral agents if the potential for
chronic adverse effects is indicated, based on:
(i) Results of this study <$ 152-40);
(ii) Results of comparable tests in § 152-30 conducted on other
species; and
(iii) The extent of expected human exposure based on residue
analysis data developed in accordance with section series 153.
(2) Data on a chronic feeding study (( 152-50) and on oncogeni-
city studies ($ 152-51) are required by 40 CTR f 4S4.16S for protozoa
if the potential for oncogenic, or other chronic adverse effects are
indicated based on:
(i) Results of this study ($ 152-40)-
(ii) Results of comparable tests in § 152-30 conducted on
other species; and
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(ill) The extent of expected human exposure based on residue
analysis data developed in accordance with section series 153.
(3) If agent replication, persistence, and the potential for
chronic adverse effects are not indicated, then no further testing
is necessary.
(e) References. Mfer to § 152-30.
152-41 Acute inhalation toxieity/infactivity study vith viral or
protozoan agents; Tier II.
(a) When required.
(1) End-us* products. Data on acute inhalation (Tier II)
are required by 40 CPU § 158.165 to support the registration of each
end-use product for which survival, replication, infectivity,
toxicity, or persistence of the microbial agent (virus or protozoa)
is observed in the test animals treated in the comparable Tier I
acute inhalation tests (i 152-32).
(2) Manufacturing-use products. Data on acute inhalation
(Tier II) are required by 40 CFR $ 158.165 to support the registration
of each manufacturing-use product for which survival, replication,
infactivity, toxicity, or persistence of the microbial agent (virus
or protozoa) is observed in the test animals treated in the compara-
ble Tier I acute inhalation tests ($ 152-32).
(b) Test standards. In addition to the applicable general
standards set forth in $ 150-3 of this subdivision and $ 80-3 of
Subdivision F, the acute inhalation study with viruses or protozoa
should be conducted in accordance vith the standards set forth in
§ 152-32 of this subdivision, with the following exceptions:
(1) Substance to be tested.
(i) The end-use product shall be tested to support the registra-
tion of an end-use product.
(ii) The manufacturing-use product shall be tested to support
the registration of a manufacturing-use product.
(2) Species.
(i) For viral agents, testing should be conducted on newly-
weaned mice and newly-weaned hamsters.
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178
(11) Tor protozoan agents, testing should be conducted on • dif-
ferent species than used In the Tier I test [§ 152-32(b)(1)]
(3) Duration of test* Test animals should be observed for 60
days, or until signs of reversible Infectlvlty subside, whichever
occurs later*
(4) Conduct of test*
(i) Observations« The animals should be observed frequently
during the day of dosing and cheeked at least once each morning
and late afternoon thereafter. Even If the animals recover com-
pletely from the exposure, the nature and onset of all gross or
visible clinical signs of Illness such as elevated temperature,
unkempt appearance, altered feeding habits, weight loss, various
forms of distress, physical depression, and other similar expres-
sions should be recorded.
(11) Sacrifice and necropsy. All test animals surviving at
termination of the observation period should be sacrificed. All
test animals, whether dying during the test or sacrificed, should
be subjected to a complete gross necropsy. If gross pathology
is observed, organs and tissues must be examined microscopically.
In addition to reporting gross pathology, microorganism dissemin-
ation, replication, and survival in animal tissues, organs, and
fluids should be determined, including survival in the intestinal
tract. Samples should be cultured on laboratory surface or other
suitable media or a host organism to provide qualitative and quanti-
tative measurements of survival and multiplication of the microor-
ganism. In addition, for tests of viral agents, samples of blood,
spleen, liver, heart, skeletal muscle, lung, and small intestine
should be examined for infectivity and be assayed by sensitive
molecular techniques, to be conducted using radioimmunoassay,
and/or enzyme-linked ianunosorbant assay, and by nucleic acid
hybridization tests.
(c) Data reporting and evaluation. In addition to the
provisions set forth in § 152-32 of this subdivision, the follow-
ing apply:
(1) ffcen using serologic and nucleic acid tests to detect
virus, the sensitivity, specificity, and limits of each assay should
be defined by comparison to a standard assay using the permissive
system.
(d) Tier progression..
(1) If replication or persistence in mammalian species is
indicated, then Tier III oncogenicity and mutagenicity tests
(§§ 152-51 and -52) may be required as specified in 40 CFR $ 158.165.
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179
(2) Certain additional studies In Tier III are required by
40 CTR 158.165 if the potential for chronic or other adverse effects
is indicated based on:
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$ 152-42 Subehronie oral doting ttudy with protozoa; Tier II.
(•) Wh«n required.
Data free subchronic oral inactivity tests are required to
support the registration of each end-use product for which there
is evidence of survival, replication, infectivity, or persistence
of the protozoan agent in the Tier X oral infectivity test
(S 152-30) and to support each manufacturing-use product which
nay legally be used to formulate such an end-use product. See 40
CPU $ 158.50 and $ 1SB.165 to deternine whether these data oust be
submitted; Section II-B of this subdivision contains an additional
discussion of the femulators' exemption and who, as a general
rule/ is responsible for submission of the required data.
(b) Test standards* The applicable general test standards
and applicable specific test standards set forth in $ 80-3 and 82(e)
through (g), respectively/ of Subdivision P shall apply, with the
following exceptions:
(1) Species. Testing should be performed in one mammalian
species, the mouse, rat, or dog.
(2) Observation of animals. In addition to the applicable
specifications set forth in § 82-1 of Subdivision F, the specifica-
tions set forth in § 152-40 of this subdivision apply.
(3) Sacrifice and necropsy. In addition to the applicable
specifications set forth in $ 82-1 of Subdivision F, the specifica-
tions set forth in $ 152-40 of this subdivision shall apply,
(c) Data reporting and evaluation. The applicable provisions
set forth in $ 82-1 of Subdivision F and $ 152-40 of this subdivision
apply.
(d) Tier progression.
M) Data on oncogenicity (§ 152-51), mutagenicity (| 152-52),
and/or chronic exposure (} 152-50) studies are required by 40 CFR
158*165 if the potential for oncogenic, mutagenic,. or other adverse
chronic effects are indicated, based on:
(i) Results of this study <$ 152*42); and
(ii) The- extent of expected human exposure based on residue
analysis data developed in accordance with section series 153.
(2) If the potential for oncogenic, mutagenic, or other
adverse chronic effects is not indicated, then no further testing is
necessary.
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.§ 152-43 Acute iptraperitoneal or intraeerebral toxicity/lnfeetivity
tests with bacteria, fungi, and protozoa; Tier II.
(a) Whan repaired. Data frost acute intraperitoneal (IP) or
intraeerebral (1C) Infeetivity tests are required by 40 CTR { 156.165
to support the registration of each end-use product for which, in
Tier I acute oral Infeetivity testing (§ 152-30), or dermal toxi-
city/infeetivity testing (§ 152-31), or intraperitoneal and Intra-
eerebral Injection testing ($ 152-33), the tast microorganism
(bacteria, fungi, or protozoa) survived for more than 2 weeks,
caused toxic effects, or caused a severe illness response in an
experimental antMT as evidenced by Irreversible gross pathology,
severe weight loss, toxemia, or death. These data are also required
to support the registration of each manufacturing-uae product
which may legally be used to formulate such an end-use product.
See 40 CTR § 158.50 and § 158.165 to determine whether these data must
be submitted; Section II-B of this subdivision contains an additional
discussion of the formulators' exemption and who, as a general rule,
is responsible for submission of the required data.
(b) Test standards. .The acute XP or 1C Infeetivity tast should
be conducted in accordance with the standards set forth in $ 152-33,
with the following exceptions:
(1) Substances to be tested, (i) The technical grade of
the active ingredient ahall be tested.
(2) Species. Two species other than those used in the compar-
able Tier I test should be tested. The preferable test species are
the rat, guinea pig, rabbit, and beagle dog.
(3) Number of animals and selection of dose levels. Test
animals, both normal and iimnunodepressed, should be Injected using
at least three graded doses of microorganisms, in sufficient numbers
to permit sacrifice at approximately two-week intervals throughout
the entire test period. At least five animals per sex, species,
dose, and state of iananodapression per sacrifice should be used.
(4) Control animals. A control group, one-half iamunode-
pressed, should be injected (I* or 1C as appropriate) with the
test vehicle containing the killed (autoclaved) tast microorganism.
Control animals should be sacrificed concurrently with the treated
animal groups. At least five animals par sax, species, and state
of iBBwnodepression should be used par sacrifice.
(5) Conduct of test, (i) Application. Approximately 0.1
ml of test solution per graded dose of test microorganism should
be injected in accordance with standard procedures and sites for
IP and 1C tests.
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182
(JJL) Duration of test. Surviving animals should be observed for
At least four month* in tests of bacterial agents and six months in
tests of fungal agents, or until th« microorganisms are no longer
detectable at the injection cite or at remote sites*
(e) Data and reporting and evaluation. In addition to the
applicable information required by f 80-4 of Subdivision T and
§ 152-33 of this subdivision, the test report should include the
following information:
(1) The LOSO for each sex for each test substance for injected
animals calculated at the end of the observation period (with method
of calculation specified) expressed in numbers of viable microor-
ganisms per kg body weight and mg product per kg body weight;
(2) The 95 percent confidence interval for the LD50; and
(3) The dose response curve and slope.
(d) Tier progression.
(1) If evidence of microbial agent replication/ persistence,
or death of test animals is observed, then the Tier ZZI chronic
feeding study (§ 152-50) is required by 40 CFR § 156.165.
(2) Data on oncogenicity and/or mutagenieity tests ($§ 152-51
and -52, respectively) are required by 40 CFR $ 158.165 if the poten-
tial for mutagenic or oncogenic effects are indicated based on:
(i) Results of this test (§ 152-43); and
(ii) The extent of expected human exposure based on residue
analysis data developed in accordance with section series 153.
(e) References. Refer to § 152-33(e).
$ 152-44 Primary dermal irritation study with microbial agents;
Tier II.
(a) When required. Data from a primary dermal irritation study
(Tier II) are required by 40 CFR $ 158.165 to support the registration
of each end-use product for which marked edema or broad erythema
was observed in the Tier I dermal irritation study {$ 152-34).
(b) Test standards. The test standards set forth in § 152-34
«PPly/ except that the use-dilution of the end-use product should
be tested.
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(c) Data reporting and evaluation. The provision* of § 152-34
apply.
(d) Tier progression. No additional tests are necessasry.
§ 152-45 Primary eye irritation study with microbial agents:
Ti«r II.
(a) When required. Data on primary eye irritation (Tier II)
are required by 40 CTR § 158.165 to support the registration of each
end-use product for which severe ocular lesions are observed in
the Tier.I primary eye irritation study (§ 152-35 of this subdivision),
(b) Test standards. The test standards set forth in $ 152-35
apply except that the use dilution of the end-use formulated product
should be tested.
(c) Data reporting and evaluation. The provisions of 5 152-35
apply.
(d) Tier progression. No additional testing is required.
§ 152-46 Effects of nicrobial pest control agents on the cellular
immune response system; Tier II.
(a) When required. Data on cellular inrane response (Tier
II} are required to support the registration of each end-use formu-
lated product for which results of the Tier I cellular immune
response test ($ 152-38 of this subdivision) indicate abnormalities
in any of the studies identified in paragraph (a) of $ 152-38 and
to support the registration of each manufacturing-use product
which may legally be used to formulate such an end-use product.
See 40 CFR § 158.50 and § 158.165 to determine whether these data
must be submitted; Section II-B of this subdivision contains an
additional discussion of the formulators' exemption and who, as a
general rule/ is responsible for submission of the required data.
(b) Test standards. [Reserved]
(c) Reporting and evaluation of data. [Reserved]
(d) Tier progression. If results of the cellular immune
response test indicate a potential hazard to humans or domestic
animals because of the survival or multiplication of the
microorganism, then appropriate Tier III tests are required by 40
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184
CTR 158.165. Specific data requirements in Tier ZZI will be deter-
mined based on the results of this test {§ 152-46) and consultation
with the Agency. Data requirements could include oncogenicity
($ 152-51), chronic feeding (J 152-50), and/or nutagenicity studies
(H 152-52).
(e) References.
(1) Refer to § 152-18(f).
(2) The following provides useful information for developing
an acceptable protocol for tests to assess antibody-forming
activity and cellular immune responses:
(i) Antibody-forming activity. At least
two groups of five mice each are treated with
the high level of test substance used in Tier
I (§ 152-18). At two tiae intervals after ex-
posure (IS and 30 days)/ test and control
animals are immunized with a standardised dose
of an antigen (sheep erythrocytes; 4 X 108
washed red blood cells). Four days later, the
animals are sacrificed and the spleen is ob-
tained. The numbers of splenic antibody plaque-
forming cells and erythrocytes are determined
by a localized hemolysis-in-gel assay using
the standard Jerne plaque assay to determine
whether there are any untoward affects on
anti-body-producing B cell competence.
The plaque assay is described below as an
example for both in vivo and in vitro tests.
Zt is not suitable merely to test the serum
antibody levels of the mice, since such test-
ing may be too insensitive to detect changes
in numbers of individual antibody producing
cells in animals already exposed to detri-
mental levels of pesticides.
(ii) Cellular immune responses. Groups
of pesticide-treated mice should be injected
with the technical preparation of the control
agent in the appropriate manner to determine
whether there has been an effect on cell-
mediated immune responses* -Jfct least one assay
for cell-mediated immunity, either with whole
animals or with cells in culture, should be
performed for animals treated with the maximum
dose of the microbial pest control agent which
elicited effects in Tier I testing (.$ 152-38).
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185
The test should be performed with groups of at
least five animals or more at 15 and 30 days
after exposure. Typical _in vivo assays would
be based on a determination of allogenic skin
graft rejection and/or resistance to highly
allogenic tumor cells.
(A) In vivo test. Groups of ten mice
each are given a full thickness allogenic skin
graft, and mean survival time is determined
and compared with the survival time of allogenic
skin grafts on untreated animals. Injection of
a tumor call Una could be performed using
mastacytoma tumor calls in which the LD50 is
readily established for control animals. It
will then be possible to determine whether the
pesticidetreated animals show a significant
enhanced susceptibility to the same numbers of
mastacytoma cells.
(B) In vitro test. Spleen cells from
sensitized with the normal allogenic
skin graft or givan tumor calls as described
in paragraph (e)(ii)(A) ara used for quantita-
tive cell-mediated immune responses in which
chromium-labeled target calls are exposed to
splenocytes from sensitized animals in vitro
in order to ascertain responsiveness of killer
T lymphocytes in the treated animal spleen.
Lymphoid cells (106) obtained from sensitized
animals - either control animals or animals
treated 15 and 30 days previously with a maxi-
mum dose of pesticide - are tested in a chromium
release assay with labeled target cells in cul-
tures • In addition, lymphoid cells from treated
animals are to be tested for their ability to
generate migration inhibitory factor jLn vitro.
For these tests, splenocytes (106) are placed
in microcapillary tubes, with or without a
specific antigen (such as the purified protein
derivative or mycobacteria if tha *«*»«T« ware
sensitized with the BCG strain of mycobacterium
or extracts of the allografts). The ability
of the mononuclear splaan cell suspensions to
•migrate from the chamber In tha presafiATttf
antigen would be- used as a correlate of cellular
immunity. As an alternative to one of the
above procedures, a blastogenic test could be
performed. Spleen cells (106) are cultured
in aicrowell chambers with and without a stimu-
lator such as phytohemaglutinin, concanavalin
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186
A, or lipopolysaccharide. The ability of the
spleen cells to respond to these mitogen* in
vitro by taking up tritiated thymidine cor-
responds to an assessment of cellular immunity.
§ 152-47 Terategenieity test vith viral agents; Tier II.
(a) When required. Data on teratogenicity studies are
required by 40 CFK $ 158*165 to support the registration of each
end-use product for which the Tier I tests on viral agents
(f 152-30 through -33 and -39) show replication of the virus in
mammalian hosts and significant damage to mammalian cells
and to support the rgistration of each manufacturing-use
product which may legally be used to formulate such an end-use
product* See 40 CTR \ 158.50 and § 158.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the formulators' exemp-
tion and who, as a general rule/ is responsible for submission
of the required data.
(b) Test standards. The applicable general test standards
and applicable specific test standards set forth in § 80-3 and
$ 83-3(e) through (g), respectively, of Subdivision P, apply/ with
the following exceptions:
(1) Substance to be tested. Testing shall be performed with
the technical grade of the active ingredient (virus).
(c) Data reporting and evaluation. The applicable provisions
of § 83-3 of Subdivision F apply.
(d) Tier progression. If evidence of teratogenic effects due
to treatment with the viruses is demonstrated/ then additional
testing in Tier III may be required by 40 CFR $ 156.165 following
consultation with the Agency.
152-48 Bacterial and fungal virulence enhancementt Tier II.
(a) When required. Data from a bacterial or fungal
virulence enhancement study are required by 40
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187
to support the registration of each manufacturing-use product
which say legally b* used to formulate such an end-use product*
See 40 CFR § 158.50 and } 158.165 to determine whether these
data must be submitted; Section II-B of this Subdivision contains
an additional discussion of the formula-tors1 exemption and who/ as
a general rule, is responsible for submission of the required
data.
(b) Test standards. Zn addition to the applicable general
standards set forth in $ 150-3 of this subdivision and $ 80-3 of
Subdivision T, bacterial or fungal virulence enhancement studies
should meet the following standards:
(1) Substance to be tested. The technical grade of the active
ingredient shall be tested.
(2) Age, sex and species. Testing should be performed on the
adult male and female laboratory mouse or hamster.
(3) Number of animals. At least 10 animals per dose level, at
least three dose levels to conduct each of three LD50 assays, and at
least 20 animals per each of five separate serial passage experiments
should be provided.
(4) Conduct of test. An initial intraperitoneal LD50 study
(expressed as numbers of microorganisms per kg body weight and mg
product per kg body weight) to determine virulence should be per-
formed. The test bacteria or fungi shall be serially passed through
mice or hamsters at least five times, in order to evaluate potential
for virulence enhancement. These steps should be followed by a
final IP LD50 to detect possible virulence enhancement, expressed
as numbers of microorganisms, per kg body weight and mg product
per leg body weight, following final passage.
(i) Initial iatraperitoneal LDSO. An IP UD50 should be per-
formed using at least three different dose levels of the test
bacteria or fungi suspended in physiological saline administered
to groups of at least 10 animals each. Animals should be observed
for mortality during a two-week period.
(ii) Serial passage. Two groups of at least 10 each should be
injected intraperitoneally separately with suspensions of the test
bacteria or fungi in physiological saline, and an adjuvant such as
Freund's adjuvant, or gastric mucin. Moribund iMnsTff and animals
surviving a two-week observation period should be sacrificed. At-
tempts to recover the test bacteria or fungi from animals dying
during testing or at terminal sacrifice should be made by culturing
samples of blood, liver, spleen, and kidneys on suitable media.
Bacteria or fungi thus recovered and identified from tissues should
be further cultured or inoculated directly following suspension in
saline or adjuvant into similar groups of untreated animals, thus
completing one serial passage. This process should be repeated
through at least five serial passages.
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188
(JLii) final intraperitoneal LDSO. IP LDSO's conducted in the
same manner as the initial IP LDSO should be performed with test
bacteria or fungi recovered from the fifth passage:
(A) An IP LDSO performed with serially-passed bacteria or fungi
suspended in saline; and
(B) An IP LDSO performed with serially-passed bacteria or fungi
suspended in an adjuvant.
(c) Data reporting and evaluation. In addition to the informa-
tion in $ 80-4 and paragraphs (b)(4)(i), (li) and (iii) of this
section, the test report should include the following information:
(1) Tabulation of response data by sex and dose level (i.e.,
number of animals showing signs of toxicity per number of animals
exposedb
(2) Time of death after dosing;
(3) IP LDSO's performed prior to serial passage, and IP LDSO's
performed following serial passage in mice (specify method of LDSO
calculation) expressed as numbers of bacteria or fungi per kg body
weight:
(i) Using inoculum suspended in physiological saline; and
(ii) Osing inoculum suspended in an adjuvant.
(4) 95 percent confidence interval for the LDSO expressed as
numbers of organisms;
(5) Dose response curve and slope;
(6) For each ani&al subjected to necropsy, the kinds of tis-
sues from which the test bacteria or fungi could be recovered and
identified per tissues cultured should be reported; and
(7) Compare number of organisms for initial and final IP LDSO
values; reduced numbers of microorganisms per kg body weight for
final IP LDSO values would Indicate increased virulence.
(d) Tier progression. (1) If evidence of virulence enhance-
ment is observed with bacterial or fungal agents them a chronic
feeding study (| 152-50) is required as specified by 40 CFR 158.165.
(2) If no evidence of virulence enhancement is observed, no
further testing is necessary.
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§ 152-49 Mammalian mutagenieity testing with microbial p«st
control agents; Tier II *
(a). When required. Data from short-term mammalian mutagen-
icity tests, as specified in $ 152-19, are required by 40 CFR 158.165
to support the registration of each end-use product that meets any
of the criteria presented in (1)-(3), balov and to support the
registration of each manufacturing-use product which may legally be
used to formulate such an end-use product. See 40 ( CTR 158.50 and
§ 158.165 to determine whether these data must be submitted; Section
II-B of this subdivision contains an additional discussion of the
formula tors' exemption and who, as a general rule, is responsible
for submission of the required data.
(1) Acute infectivity tests are positive in Tier I stadies
(as specified in 5$ 152-30 through -33 of this subdivision);
(2} Adverse cellular effects are observed in cellular immune
response studies (as specified in Tier I § 152-38); or,
(3) Positive results are obtained in tissue culture tests
with viral agents (as specified in J 152-39).
(b) Test standards. Tests should be performed according to
both the general and specific standards set forth in $ 152-19 of
this subdivision, with any necessary modifications due only to the
nature of the test substance.
(c) Test substance. The technical form of the active ingre-
dient shall be tested. In addition, the purest infective form
(PIP) .shall be tested for viral agents.
(d) Reporting. The information to be reported is the same
as that specified in § 152-19 of this subdivision with any necessary
modifications due to the nature of the test substance.
(e) Tier progression. Positive results for mutagenicity in any
cell or organism test will require additional mutagenicity
testing in Tier III (§ 152-52) and/or oncogenicity testing (§ 152-
51) as specified in 40 CFR § 158.165. The specific test required in
Tier XII shall be determined after consultation with the Agency.
(f) References. Refer to $ 152-19.
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190
Group B-3: Tier III Testing.
§ 152-50 Chronic feeding study; Tier III.
(a) When required. Data on a chronic feeding study are re-
quired by 40 CFR § 158.165 to support the registration of each end-use
product for which the potential for chronic adverse effects (e.g.,
replication or persistence of viral or subviral constituents,
protozoans, fungi/ or bacteria) are demonstrated by any of the
following Tier IZ tests: §( 152-40 through -43, -46, and -49.
These data are also required to support the registration of each
manufacturing-use product which «ay legally be used to formulate
such an end-use product. Consultation with the Agency is advised
before performing these studies. See 40 CFR $ 158.50 and { 158.165
to determine whether these data must be submitted; Section ZI-B of
this subdivision contains an additional discussion of the formula-
tors' exemption and who, as a general rule, is responsible for
submission of the required data.
(b) Combined testing. A chronic feeding study may be combined
with an oncogenic evaluation pursuant to $ 152-51, provided that
standards for both types of testing are met.
(c) Test standards. The applicable general and specific
standards set forth in H 80-3 and 83 (d) and (e), respectively,
of Subdivision F apply, with any necessary modifications due to
the nature of the test substance.
(d) Data reporting and evaluation. The provisions set forth
in 5 83-1 of Subdivision F shall apply, with any necessary modifi-
cations due to the nature of the test substance.
$ 152-51 Oneegenicity studies; Tier III.
(a) When required. Data from oneogenieity testing may be
required by 40 CFR § 158.165 to support the registration of each
end-use product for which the potential for oncogenic effects is
indicated (e.g., adverse cellular effects due to presence, replica-
tion, or persistence of viral or subviral constituents,-or bacteria,
fungi or protozoans; or mutagenic effects) by any of the following
Tier II tests: §§ 152-40 through -43, -46, and -49. These data
are also required to support the registration of each manufacturing-
use product which may legally be used to formulate such an end-use
product. Consultation with the Agency is advised before performing
these studies. See 40 CFR $ 158.50 and § 158.165 to determine whether
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191
these data oust be submitted; Section II-B of this subdivision
contains an additional discussion of the formula ton* exemption
and who, as a general rule, is responsible for submission of the
required data.
(b) Combined testing. A chronic feeding study may be combined
with an oncogenic evaluation pursuant to § 152-50, provided that
standards for both types of testing are Bet.
(c) 'Test standards. The applicable general and specific
standards set forth in j$ 80-3 and 83(d) and (e), respectively/ of
Subdivision F apply, with any necessary modifications due to
the nature of the test substance.
(d) Data reporting and evaluation. The provisions set forth
in $ 83-2 of Subdivision P shall apply, with any necessary modifica-
tions due to the nature of the test substance.
$ 152-52 Mtttagenieity testing; Tier III-.
(a) When required. Data from whole animal mutagenicity test-
ing may be required by 40 CFR § 158.165 to support the registration
of each end-use product for which the potential for mutagenic effects
is indicated (e.g., adverse cellular effects due to presence, repli-
cation, or persistence of viral or subviral constituents, bacteria,
fungi or protozoa) by any of the following Tier II tests: H 152-40
through -43, and -46 or -49. These data are also required to
support the registration of each manufacturing-use product which
may legally be used to formulate such an end-use product. Consul-
tation with the Agency is advised before performing this testing.
See 40 CFR § 158.50 and § 158.165 to determine whether these data must be
submitted; Section II-B of this subdivision contains an additional
discussion of the formulators' exemption and who, as a general rule,
is responsible for submission of the required data.
(b) Test standards. The applicable general and specific
standards set forth in H 80-3 and 84-1 through -4 of Subdivision F
apply, with any necessary modifications due to the nature of the
test substance.
(c) Data reporting and evaluation. The provisions set forth
in H 84-1 through -4 of Subdivision F apply, with any necessary
modifications due to the nature of the test substance.
(d) References. Refer to § 84-5 of Subdivision F.
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192
§ 152-53 Teratogenieity etudies; Tier III.
(a) When required. Data on teratogenicity studies ar« re-
quired by 40 CFR § 158*165 to support the registration of each end-use
product for which the potential fox teratogenic effects is expected
based on the presence or persistence of fungi, bacteria, viruses,
or protozoa in manualian species as a result of testing perforaed
in Tier II (e.g., f§ 152-40 through -43, -46, -48, and -49). These
data are also required to support the registration of each manufac-
turing-use product which Bay legally be used to formulate such an
end-use product. Consultation with the Agency is advised before
performing the** studies. See 40 CTR § 158.50 and § 158.165 to
determine whether these data Bust be submitted; Section ZI-B of
this subdivision contains an additional discussion of the formula-
tor*' exenption and who, as a general rule, is responsible for
submission of the required data.
(b) Test standards. The applicable general and specific
standards set forth in §§ 80-3 and 83-3(e) through (g), respec-
tively, of Subdivision F apply, with any necessary modifi-
cations due to the nature of the test substance.
(c) Data reporting and evaluation. The provisions set forth
in J 83-3 of Subdivision F apply, with any necessary modifica-
tions due to the nature of the test substance.
Series 153: RESIDUE ANALYSIS GUIDELINES FOR BIORATIONAL PESTICIDES
§ 153-1 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 § 158.165 in connection with each applica-
tion for registration of a biorational pesticide product where
usage may result in residues in or on food for humans or feed for
domestic animals used for human food. This petition must contain
data satisfying the requirements of 40 CPU § 158.165 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 magni-
tude of residues in or_jpn 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 carryover of residues into meat, milk, poultry,
and eggs.
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193
(c) Authority. Pesticides intended for use en food or feed
crop*, or where usage m«y 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 Pood, Drug, and Cosmetic
Act CfTDCA" 21 U.S.C. 346, 346a and 348). The procedural regula-
tions for filing petitions for a tolerance or an exemption are In-
cluded in 40 CTR § 180.7.
(d) Location of guidelines. { 153-3 lists the guidelines
for biochemical pest control agents and ( 153-4 refers to guidelines
for microbial pest control agents*
$ 153-2 [Reserved]
153-3 Residue data guidelines for biochemical pest control
agents.
(a) When required. (1) A petition for a tolerance or for an
exemption from the requirement of a tolerance is required by 40
CTR § 158.165 in connection with each application for the registration
of each end-use product and manufacturing-use product composed of or
containing a biochemical when the following conditions are met:
(i) The product is intended for use on food or feed crops or its
use is expected to result in residues in or on food or feed; and
(ii) The rate of application exceeds 0.7 ounces (20 grams) of the
biochemical (active ingredient) per acre per application.
(2) Residue data requirements will be determined on a case-by-
case basis for blochemlcals applied directly to food or feed and for
biochemicals with application rates not expressable in ounces per
acre per application.
(3) 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 or feed crops or for uses expected to result
in residues in or on food or feed, when the following conditions are
met:
(i) Toxicology data developed from Tier I testing in accordance
with $$ 152-10 through -18 of this subdivision indicate that testing
at Tier II is not requirear and
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194
.(11) *h« *ate of application is equal to or less than 20 grass
of biochemical (active ingredient) par acre per application.
(b) Procedures, standards, and reporting* (1) General, in
addition-to the applicable general provisions of H 150-3 and -4,
the information provided in Subdivision 0 (Series 170} should be used
as general guidelines for test procedures, test standards, and the
reporting of data.
(2) Specific aspects, (i) The full range of residue chemistry
data detailed in Subdivision O (Series 170) apply to products for.
which:
(A) Toxicity testing proceeds to Tier IX or III, as described in
H 152-19 through -29 of this subdivision/ or
(B) The application rate exceeds 0.7 ounces (20 .grams) active
ingredient per acre per application.
(ii) A petition for a tolerance or an exemption from the require-
ments of a tolerance shall be required in connection with each appli-
cation for registration for each biochemical that meet* either of
the criteria set forth in paragraph (b)(2)(i)(A) or (B) of this
section.
5 153-4 Besidue data requirements for microbial pest control agents.
(a) When required. Residue data are required by 40 CFK § 158.165
to be included in a petition for a tolerance or for an exemption
from the requirement of a tolerance, in connection with each appl-
ication for registration of a manufacturing-use product or end-use
product composed of or containing a microbial agent, when the fol-
lowing 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 X toxicology studies conducted in
accordance with if 152-30 through -39 of this suMivLslon indicate
that Tier XX toxicology studies described in f§ 152-40 through -48
are required.
(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 or feed crops or for uses expected to
result in residues in or on food or feed, when the toxicology data
developed from Tier X testing, in accordance with $$ 152-30 through - 39
of this subdivision, indicate that testing at Tier XI is not required.
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195
(b) Procedures, standards, and reporting. In addition to th«
provisions set forth in 55 150-3 and -4 that are applicable/ the
following guidance is provided for conducting, developing, and report-
ing the residue data that the Agency requires to support a petition
for a tolerance or for an exemption froo the requirement of a
tolerance:
(1) Subdivision 0 (Series 170) contains applicable information.
For the qualification of viable bacteria or fungi (or their spore
forms), artificial stadia Bay be employed following washing/ hoaogeni-
zation, centrifugation, or other appropriate treatment* to collect
and/or concentrate the organism. With viruses and protozoa, specific
hosts may be required for quantitative measurements of viable resi-
dues. These recovery techniques may be supplemented or even replaced
by appropriate proven techniques involving fluorescent antibodies
or electron microscopy*
(2) Discussion with appropriate Agency scientists may be helpful
before steps are taken to develop residue data of the nature outlined
in this section.
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Series 154: NONTARGET ORGANISM HAZARD GUIDELINES FOR
BIORATIONAL PESTICIDES
$ 154-1 General information.
(a) Scope of guidelines for nontarget organisms. Section
series 154 outlines the Agency guidelines for the submission of
data and information relating to pesticidal effects on terrestrial
wildlife, aquatic animals, plants/ and beneficial insects in support
of applications for registration of naturally occurring or synthetic
biochemical pest control agents and microbial pest control agents.
In many instances these guidelines refer to other subdivisions per-
taining to conventional pesticide products.
(b) Approach, the tests to evaluate pesticidal effects on
terrestrial animals, aquatic animals, plants, and beneficial
insects are arranged in a hierarchical or tier system, beginning
with acute testing at Tier I. Tier II testing involves Environ-
mental Fate testing for biochemical agents and Environmental
Expression testing for microbial agents (section series 155) to
estimate environmental concentrations of pesticides after applica-
tion. Tier III consists of further acute, subacute, and chronic
laboratory testing on nontarget organisms, and Tier IV consists of
applied field tests encompassing both nontarget organisms and
environmental fate or expression. The results of each tier of
tests must be evaluated to determine whether further testing is
necessary. It is expected that the extent of testing will diminish
with each subsequent tier. (Figures 1 through 5 illustrate the
tier testing systems for biochemicals for terrestrial wildlife,
aquatic animals, plants, and terrestrial and aquatic insects,
respectively. Figures 6 through 10 illustrate the tier testing
systems for microbial pest control agents for terrestrial wildlife,
aquatic animals, plants, and terrestrial and aquatic insects,
respectively.)
(c) Organization. The guidelines are divided into two
broad categories - those for biochemicals and those for microbial
pest control agents. The protocols to develop the data are
organized in a tier arrangement within each of these categories
and each tier contains testing protocols pertaining to the major
groups of'nontarget organisms (terrestrial wildlife, aquatic
animals, plants and insects). Table 6 illustrates this organiza-
tion.
$§ 154-2 through -5 [Reserved].
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197
Figure ^--TERRESTRIAL WILDLIFE TI£R TCSTQiG SCHEME FOR BIOCHEMICAL
PESTICIDES
QOESnOH; It the biochemical highly
volatile - (estimated volatility
> Sx10"5 ata. m3/aol.)7
YES
THE FOLLOWING TEST
IS INDICATED >
1) Avian Single
Dose Oral
Toxicity Test,
S 154-6.
MO
I
V
THE FOLLOWIKC TESTS
ARE INDICATED;
1) Avian Single Dose
Oral Toxicity Test,
§ 154-6.
2) Avian Dietary Toxicity
Test, | 154-7.
Consider use pattern,
available fate data, and
human safety data
TIER II
arVIRON
FATE TESTING
Potential adverse
effects observed
Environmental
Fate Testing,
i series 155
Mo potential
adverse effects:
no further
testing necessary
I
V
Fate characteristics
indicate exposure
I
I
v
I
V
Fate loharecteristics do
not indicate exposure: no
further testing necessary
TIER III
SFFECTS TESTING
Conduct testing in
accordance with
Subdivision E,
5 154-12
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198
Figure 2~ AQUATIC ANIMAL TIER TESTING SCHEME FOR BIOCHEMICAL PESTICIDES
The biochemical is not introduced
directly into an aquatic environ-
ment when used as directed
I
V
1 • Does use pattern or formulation
preclude significant exposure of
aquatic environment, under normal
use conditions? on
2. Is the amount applied ••all (£0*7
oz. A. I. /Acre (20 grams/acre)? or;
3. Is the estimated volatility £
5x1 0'5 atm. mVmol? "
The biochemical is introduced
directly into an aquatic environ-
sent when used as directed
I
7
Conduct testing as indicated in
Subdivision E of the guidelines,
$§ 72-1 through -7
I
TIER IV
If answer is YES to one or more,
conduct the following
tests:
1. Fish acute bioassay
(§ 154-8)
2. Aquatic invertebrate acute
bioassay ($ 154-9)
I
V
No potential adverse
effects: no further
testing necessary
V
If answer is NO to all three,
conduct both effects (TIER I)
and fate (TIER II) tests:
1. Fish acute bioassay
(§ 154-8)
2. Aquatic invertebrate acute
bioassay (§ 154-9)
I
V
Potential adverse
effects observed
TIER II
Conduct environmental
fate testing <§ series 155)
I
V
Fate characteristics
do not indicate
aquatic exposure*
no further
testing necessary
Fate characteristics
indicate aquatic
exposure
TIER III
Conduct testing as
indicated in
§ 154-13
-------
199
Figure ..3—NONTARGET PLANT TIER TESTING SCHEME TOR BIOCHEMICAL PEST
CONTROL AGENTS.
TIER I. Consider the mode of action and use:
(Tests to be conducted on a case-by-case bails as specified in
40 CFR § 158.165)
Used to control plant
growth and development
I
Used to control animals
(including insects)
I
I I 7V
V V General broadcast Bait or other
Test: Target area Test: Nontarget area (not contained) containers:
phytotoxicity, phytotoxicity, § 154-10 I no testing
§ 154-10
To be used at levels
exceeding 3x the
highest level of
naturally occurring
biochemical
I necessary
I
To be used at levels
up to 3x the highest
level of the naturally
occurring biochemical
I
V
Synthetic or not used
within plant community
type from which derived
I
V
Terrestrial use;
Test: Terrestrial
phytotoxicity,
§ 154-10
I
I
V
Naturally
occurring;
no testing
necessary
I
V
Aquatic use;
Test: Aquatic
phytotoxicity,
§ 154-10
I
TIER II
Adverse effects
No effects; no further testing
Environmental fate testing <$ series 155)
I
Determine movement from site of application:
Movement by soil, water, or air
No movement anticipated;
no further testing
v
TIER III Conduct aquatic and terrestrial phytotoxicity testing, § 154-14
-------
200
-Figure -4—NONTARGET INSECT TESTING SCHEME FOR BIOCHEMICAL PESTICIDES
TERRESTRIAL INSECTS
TIE* I
Adverse effects noted during efficacy
testing, end/or other data exist
to indicate potential for adverse
effects on nontarget insects
I I
YES NO
TIER II
Environmental fate testing No testing
($ series 155) necessary
Fate testing indicates potential for exposure.
I I
] I
V V
YES NO
TIER III | I
V V
Consult with the Agency prior to Bo further testing
testing. Type of data will depend
on the nature of adverse effect
noted (i 154-15)
-------
201
Figure 5—NONTARCET INSECT TESTING -SCHEME FOR BIOCHEMICAL PESTICIDES -
AQUATIC INSECTS
The biochemical is not
introduced directly into an
aquatic environment when
used as directed
The biochemical is introduced
directly into an aquatic
environment when u»ed a*
directed
I
TIER I Test according to the
aquatic animal tier
tasting scheme for
biochemical pesticides,
(§ 154-9)
Conduct testing as indicated
in by Subdivision* E and
L of the guidelines
I
TIER II
Potential adverse effects observed
Conduct environmental fate
testing (§ series 155}
I
No potential adverse effects;
no further testing necessary
I
V
Fate characteristics indicate
aquatic exposure
TIER III Conduct testing as indicated in
Subdivisions E and L of
the guidelines
Fate characteristics do not
indicate aquatic exposure; no
further testing necessary
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202
Figur* 6—TERRESTRIAL WILDLIFE TIER TESTING SCHEME FOR MICROSIAL
PEST CONTROL AGENTS
Non-genetically-engineered
microorganisms
I
I
V
TIER I I) The following
EFFECTS test* are.
TESTING indicated
1) Avian single dose oral toxicity
and pathogenieity test (§ 154-16)
2) Avian injection pathogenicity
tests (§ 154-17)
3) Wild mammal single dose oral
toxicity and pathogenieity
testing ($ 154-18), would be
indicated only if data required
for human safety are not
sufficient to assess potential
effects to wild mammals
Genetically-engineered
microorganisms
II) The tests in TIER I are
indicated plus TIER II
environmental
expression testing
I
Consider use pattern, any
available expression data
and human safety data
Consider use pattern, any
available expression data
and human safety data
Ho potential adverse
effects; no further
testing necessary
Toxic and/or pathogenic
effects
Potential adverse
effects
TIER II Environmental expression
ENVIRONMENTAL testing, § series 155
EXPRESSION |
TESTING I j
I
V
Expression characteristics
indicate exposure
Expression characteristics do
not indicate exposure; no
further testing necessary
-------
203
Figure 6~(continued)
TIER III Type of effect(s)
EFFECTS from TIER 1 testing
TESTING |
V V
Pathogenic effects Toxic effects
I I
I I
V V
Long tern avian pathogenicity Conduct testing according
and reproduction test* to Subdivision E and proceed
J 154-26 with tier testing as indicated
in Subdivision E; see $ 154-25
Mo adverse effects;
no further testing
V necessary
Pathogenic effects observed
V
TIER IV The applicant should re-consider the application
EFFECTS AND for registration. Pathogenic effects at TIER III
EXPRESSION and beyond raise serious questions concerning the
TESTING registration of any nicrobial pest control agent.
The Agency will review all data and decide if a
decision concerning registration should be made
without further testing.
I
I
7
Conduct simulated and actual
field testing for manuals
and birds (| 154-33) pending
prior Agency review
-------
204
Figure 7—AQUATIC ANIMAL TIER TESTING SCHEME FOR MICROSIAL PEST
CONTROL AGENTS
Non-genetically-engineered
microorganisms
Genetically-engineered
microorganisms
Conduct toxicity/pathogenieity tests
as follow*t
TIER I
Terrestrial Application
Conduct the appropriate TIER I
te«ta, plus TIER II
environmental expression tests
1. Freshwater fish toxieity/pathogenicity test -
one species (§ 154-19)
2. Freshwater aquatic invertebrate toxicity/-
pathogenicity test - one species (§ 154-20)
Direct application to fresh water
1. Freshwater fish toxieity/pathogenicity test -
two species (§ 154-19);
2. Freshwater aquatic invertebrate toxicity/-
pathogenicity test - two species (§ 154-20)
Direct application to marine or estuarine waters
1. Estuarine fi marine animal toxieity/pathogenicity
test - one fish species and one invertebrate
species (§ 154-21)
2. Freshwater fish toxieity/pathogenicity test -
one species ($ 154-19)
3. Freshwater aquatic invertebrate toxicity/
pathogenicity test - one species (§ 154-20)
I I
i I
V V
No potential Potential
adverse effects advene effects
observed observed
I.
Do host spectrum or beneficial
insect tests indicate a broad
host spectrum such that suscep-
tibility of aquatic invertebrates
is likely?
-------
205
Figure "7~( continued)
I I I _
V V
If NO, then no further If YES, either-.
testing is necessary 1. Test additional aquatic
_______________ invertebrates (§ 154-20);
I I 2. Conduct TIER II testing
I | (f series 155)
v I
Additional species tasted, !-> Additional species tested,
no potential adverse effects; potential adverse effects
no further testing necessary observed |
V
TIER II Conduct environmental
____________________ «xpression testing
I (§ series 155)
I I
V V
Ho exposure indicated; _ Environmental expression
no further testing I analysis indicates
necessary I exposure
V
_______ Further testing
I indicated TIER III |
V V
TIER III Pathogenic, or pathogenic and toxic effects: Toxic effects:
1. Definitive aquatic animal pathogenicity Conduct testing according
tests ($ 154-27)7 or to Subdivision E, and
proceed with tier testing
2. Embryolarvae and life cycle tests as indicated in Sub*
(§ 154-28); or division E (see § 154-25)
3. Aquatic ecosystem-pathogenic!ty tests
(§ 154-29); or
4. Special tests: tissue culture, micro- _________________
organism/stress interaction tests I
<$ 154-30) I
I I
V V
No adverse effects; Adverse effects
no further testing observed
necessary I
I
V
TIER IV Simulated or actual field
tests of aquatic animals
Uee note, below) (§ 154-34)
NOTE; Pathogenic effects at TIER III and beyond raise serious questions
concerning registration. The Agency will review all data before TIER V
testing and decide if a decision concerning registration should be made
without further testing.
-------
206
Figure 8—HONTAKGET PLAKT TIER TESTING SCHEME FOR MICROBIAL PEST
CONTROL AGENTS
TIER I.
Consider the use and node of Action:
Used to control plant
growth and developaent
I
Used to control
animals
I
Test: Target area
pbytotoxicity,
§ 154-22
Test: Nontarget area
phytotoxicity
Agent not
occurring in
area to be used
Agent naturally
occurring in area
to be used
I
Used at level
exceeding
natural occurrence
I
V
To be used at levels
below or similar to
natural occurrence;
no testing necessary
Terrestrial Dse
Test: Terrestrial
phytotoxicity,
| 154-22
Aquatic use
Test: Aquatic
phytotoxicity,
§ 154-22
TIER II
Adyerse V
effects No effects; no further testing necessary
I
Environmental expression testing, § series 155
I
Determine movement from site of application:
Movement in soil, water, or air
No movement anticipated;
no further testing necessary
Adverse effects on terrestrial plants
are indicated in TIER I, and terrestrial
exposure is indicated in TIER II testing
I
V
TIER III Conduct terrestrial
phytotoxicity testing
(5 154-31)
Adverse effects on aquatic plants ar>
indicated in TIER X, and aquatic exp<
sure is indicated in TIER II testing
Conduct aquatic
phytotoxicity testing
( 154-31)
-------
207
Figure 9—NONTARGET INSECT TIER TESTING SCHEME FOR KICRO8IAL PEST
CONTROL AGENTS: TERRESTRIAL INSECT TESTING WITH
NON-GENETICALLY-ESGINEERE0 MICROORGANISMS
TIER I Test:
1. Honey Bee toxicity and pathogenicity test ($ 154-24); and
2. Toxicity and pathogenicity tests on three other species of
beneficial insects from the following groups (no acre than
one species per group):
Predaceoue hemipterans (§ 154-23)
Predaceous coleopterans (\ 154-23)
Predaceous mites (f 154-23)
Predaceous neuropteraas (} 154-23)
Parasitic hymenopterans (f 154-23)
I
V
Consider Tier I test results, use pattern,
available research and development data on
specificity, available fate information
I I
I I
V V
Potential toxic and/or No potential adverse
pathogenic effects effects observed;
I no further testing
I necessary
I
V
TIER II Environmental expression testing
indicated {§ series 155)
I I
I V
I Expression characteristics
! indicate exposure
I I
V V
Expression characteristics Consider the type of adverse effecc
do not indicate exposure determined from TIER I testing
I I I
I I I
I V V
7 Pathogenic effects Toxic effecrs
Mo further testing necessary I I
I I
I I
V V
TIER III Consult with Agency Consult with Agency
regarding simulated or regarding possible use
actual field testing restrictions, label
(J§ 154-35 and -36) precautions, or further
or other testing testing specific
specific to the problem to the problem
-------
208
10—NONTARGET INSECT TIER TESTING SCHEME FOR MICROBIAL PEST CONTROL
AGENTS: TERRESTRIAL INSECT TESTING WITH GENETICALLY-ENGINEERED
MICROORGANISMS
TIERS I AND II
Conduct appropriate TIER I tests, plus
TIER II environmental expression tests
I
I
I
V
Consider use pattern, and available research
and development data on host range
I I
I I
v I
No potential adverse effects; V
no further testing necessary Potential adverse effects indicated
I I
I V
| Expression characteristics
[ do not indicate exposure;
| no further testing necessary
V
Expression characteristics
indicate exposure
I
I
I
V
Type of adverse effect
determined from TIER I testing
I I
I I
I I
V V
Pathogenic effects Toxic effects
I I
I I
TIER III ! I
I V
| Consult with Agency regarding
V possible use restrictions/ label
Consult with Agency regarding precautions, or further testing
simulated or actual field specific to the problem
testing ($ 154-35 and -36),
or other testing specific to
the problem
-------
209
Table 6— SUMMARY OP NONTAKGET HAZARD GUIDELINKS BY PESTICIDE TYPE, TIER, AND NON-TARGET ORGANISM GIWMJP
Terrestrial Wildlife Aquatic Animals
Plants
Beneficial Insects
(1) Biochemical i
TIER 1
TIER II
f| 154-6. -7 and ff 154-8 and -9
f 71-3 (Subdivision E)
TIER 111 f 154-12
{ 154-10 and
( 122-1 and -2
(Subdivision J)
154-11
Environmental fate tests (f| 155-1 through -14)
f 154-13
f 154-14
f 154-15
(2) Hicrobials
TIER I
TIER II
ff 154-16, -17,
and -18
i 154-19, -20. { 154-22 f 154-23 and -24 I
and -21
Environmental expression tests (see f| 155-15 through -23)-——-—
TIER III ff 154-25 and -26
TIER IV f 154-33
f 154-25 and
-27 through -30
| 154-34
f 154-31
None
None
f 154-35 and -36
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210
Subserves 154A: BIOCHEMICAL AGENTS
Group .A-1: Ti«r I Testing.
| 154-6 Avian single dose oral toxieity test; Ti«r I.
(a) Wh«a required. Data on the avian acute oral toxieity of a
biochemical pesticide are required by 40 CFR § 156.165 to support
the registration of each end-use product intended for outdoor appli-
cation and each oanufacturing-use product that may legally be used
to formulate such an end-use product* See 40 CFR § 158*50 and
$ 158.165 to determine whether these data oust be submitted; Section
II-B of this subdivision contains an additional discussion of the
formulators' exemption and who, as a general rule, is responsible
for submission of the required data.
(b) Test standards* The data should satisfy the general test
standards in $ 70-3 of Subdivision E and all of the following test
standards:
(1) Test substance* The technical grade of each active Ingre-
dient in the product shall be tested.
(2) Species * Testing should be performed on one avian species
(preferably bobwhite quail or mallard duck)* The species selected
should be the same as that selected for the avian dietary toxieity
test in 5 154-7.
(3) Age. Birds used in this test should be at least 16 weeks
old at.the beginning of the testing period. Within a given test,
all birds should be of the sane age.
(4) Controls. A concurrent control group is recommended. If a
vehicle (carrier, solvent, or diluent) is used, the concurrent control
group should be treated with the vehicle* Vehicles known to be
toxic, synergistic, or antagonistic should not be used.
(5) Sumber of birds per dosage level. Each treatment and con-
trol group should contain at least 10 birds. When ^w^Jj^one treatment
group is tested, at least 30 birds should be tested at'that dosage
level.
-------
211
(6) Determination of LDSO. The test data must establish:
(i) That the avian single-dose oral LDSO is greater than a maximum
test dosage, £ mg/kg, where jc is determined by the following equation:
maximum application rate in grass
x ag/kg • 2000 ag/kg x active ingredient per acre
" 454 g/acre*
Only one concorrent vehicle control group and one treatment group
dosed at x ag/kg say be necessary; or
(ii) A. precise LDSO value and corresponding 95 percent
confidence interval.
(c) Reporting and evaluation of data* The requirements of
§ 70-4 and § 71-1(c) of Subdivision E apply for all products to be
tested in accordance with this section.
(d) Tier progression. {1) Testing at Tier XI is required by
40 CFR § 153.165 if any one or more of the following occur:
(i) The maximum expected environmental concentration is equal
to or greater than 1/5 the avian single-dose oral LDSO value
expressed in ppm. The LDSO in mg/kg is converted to ppm by the
following formula:
ppm • LDSO T average daily food consumption (g)
body weight (g)
(ii) Signs of abnormal behavior are observed in the avian
singledose oral toxicity test at levels equal to or less than the
maximum expected environmental concentration)
(iii) Growth, development, or reproductive effects may be
expected, based on observed effects in the avian singledose oral
toxicity test, available environmental fate data, use pattern
information, and results of tests required to support human safety
(Subdivision F).
(2) If none of the criteria in paragraph (d)(1) of this
section are met, then additional testing at Tier II [environmental
fate testing (§5 155-1 through -14)] is not necessarv.
(e) Refertnce; test protocol. An example of an acceptable
protocol for conducting an avian single-dose oral toxicity test is
provided in $ 71-1 of Subdivision E.
*(Number of grams per pound. One pound is a typical application rate
for conventional pesticides.)
-------
212
154-7 Avian dietary toxicity teat: Tier 1.
(a) When required. Data on the avian dietary toxicity of a
biochemical pesticide are required to support the r«9istration of
•aeb end-use product that ia not highly volatile (estimated volatility
greater than 5x1O"5 atm. sP/sol.) and that ia intandad for outdoor
application, and each manufacturing-use product that may ba uaad to
formulate such an end-use product* S«a 40 CTR § 158.50 and § 156.165
to determine whether thaaa data mist ba submitted; Saction II-B of
thia subdivision containa aa additional discusaion of tha formulators1
axaaption and who/ aa a general rule, ia responsible for submission
of the required data.
(b) Test standards. Data should satisfy the general test
standards in $ 70*3 of Subdivision B and all of the following
test standards:
(1) Test substance. The technical grade of each active
ingredient ia the product shall be tested.
(2) Species. Testing should be performed on one avian species
(preferably bobwhite quail or mallard duck). The species selected
should be the same as that selected for the avian single-dose oral
toxicity test required by $ 154-6.
(3) Age. Birds used in this study should be from 10 to 17
days old at the beginning of the testing period. Within a given
test, all birds should be of the same age.
(4) Controls. A concurrent control group is required. If
a vehicle (carrier, solvent, or diluent) is used, the concurrent
control group should be treated with the vehicle. Vehicles known to
be toxic, synergistic, or antagonistic should not be used.
(5) Number of birds per concentration level. Each treatment
and control group should contain at least 10 birds. When only one
treatment group is tested, at least 30 birds should be tested at
that treatment level.
(6) Determination of LC50. the test data must establish:
(i) That the 8 day dietary (5 days treated diet and 3 days
untreated diet) LCSO ia greater than a maximum test concentration,
£ ppm, where £ ppa is determined by the following equation:
naximum application rate in grams
x ppm • 5000 ppm x active ingredient per acre
454/acre*
•Number of grams per pound. One pound is a typical application
rate for conventional pes,t,i=ldes.
-------
213
Only en* concurrent vehicle control group and on* treatment group
fed a concentration equal to it ppm may b* necessary> or
(ii) A precise LD50 value and corresponding 95 percent
confidence intervals.
(c) Reporting and evaluation of data. The provisions of
§ 70-4 and § 7l-2(c) of Subdivision E apply for all products to be
tested in accordance with this section.
(d). Tier progression. (1) Testing at Tier II is required by
40 CFR § 158.165 if any one or more of the following occur:
(i) The maximum expected environmental concentration is equal
to or greater than 1/5 the avian dietary I/CSQ value
(ii) Signs of abnormal behavior are observed in the avian
dietary toxieity test at levels equal to or less than the maximum
expected environmental concentration;
(iii) Growth/ development, or reproductive effects may be
expected, based on observed effects in the avian dietary toxieity
test, available fate data, use pattern information, or results of
tests required to support human safety (Subdivision F).
(2) Zf none of the criteria in paragraph (d)(1) of this
section is met, then additional testing at Tier II, Environmental
Pate Testing {§§ 155-1 through -14), is not necessary.
(e) Reference; test protocol. An example of an acceptable
protocol for conducting an avian dietary toxieity test is provided
in § 71-2 of Subdivision E and as required by 40 CFR 158.145.
§ 154-8 Freshwater fish acute bioassayt Tier I.
(a) When required. (1) Toxieity (or toxic-like effects)
data on a freshwater fish are required by 40 CFR § 158.165 to
support the registration of each end-use product intended for
outdoor terrestrial application and each manufacturing-use product
that legally may be used to formulate such an end-use product.
See 40 CFR § 158.50 and J 158.165 to determine whether^thes* data
must b* submitted; Section II-B of this subdivision contains an
additional discussion of the .formulators1 exemption and who, as a
general rule, is responsible for submission of the required data.
(2) If the pesticide will be introduced directly into an
aquatic environment when used as directed, then it must be tested
as indicated in §§ 72-1 through -7 of Subdivision E, and as
required by 40 CFR § 158.145.
-------
214
(b) Test ^standards. The general and specific test standards
for biochemical pesticide* are the sane as those set forth in
Subdivision E in §f 70-3 »n(i 72-l, respectively, with the following
exceptions:
(1) Testing should be performed on one species, preferably
rainbow trout; and
(2) One maximum hazard concentration may be tested in lieu of
conducting a definitive IC$Q or £€50 test if exposure of 30 organism*
to a concentration of 1/000 tines the expected aquatic environmental
concentration or 100 mg/1 of water (whichever is greater) produces
less than 50 percent mortality*
(e) Reporting of data. The reporting provisions are the
same as those set forth in Subdivision £ of the guidelines
(| 72-1).
(d) Tier progression* (1) Biochemical agents that meet one
of the following three criteria will not require testing at Tier II
[environmental fate (H 155-1 through -14)1 except as noted in
paragraph (d)(2) below:
(ii) The amount of biochemical applied is small (less than 0.7
ounces (20 grams) active ingredient per acre per application); or
(iii) The estimated volatility of the biochemical is high
(equal to or greater than 5x10~5 ata. mVmol).
(2) Biochemical agents that meet one or more of the above
three criteria of paragraph (d)(1) of this section require testing
at Tier II [environmental fate (§§ 155-1 through -14}] if any of
the following occur:
(i) Signs of abnormal behavior are reported in the Tier I
test at concentrations less than or equal to the maximum expected
concentration in water; or
(ii) The maximum expected concentration in water is equal to
or greater than 0.1 the LCgo determined by testing outlined din this
section; or
(iii) The maximum expected concentration in water is »qual to
or greater than 0.01 the LCjQ determined by testing ewtlined in
this section end adverse effects on growth/ develdevelopment, or
reproduction may be expected, based on Tier I test data (§ series
154), available environmental fate data (e.g., from the product's
research and development)/ use pattern information, or available
effects data on phylogenetically similar target species.
-------
215
(3) If the criteria in paragraph (d)(J) of this section are
not met, then additional testing at Tier II (§§ 155-1 through -14)
is required.
(e) References. (1) Examples of acceptable protocols for
conducting a freshwater fish acute toxicity study stay be found in
the following references. Fish species listed in reference (ii)
are acceptable, with the exception of goldfish:
(i) ASTH Standard £ 729-80, Practice for Conducting Acute
Toxicity Tests with Pishes, Kacroinvertebrates, and Amphibians.
American Society for Testing and Materials, 1916 Race Street,
Philadelphia, PA 19103.
(ii) 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.
(2) The following may contain useful background information
for developing acceptable protocols:
(i) Weber, C.E. (ed.). 1973. Biological field and laboratory
methods for measuring the quality of surface waters and effluents.
U.S. Environmental Protection Agency, Environ. Monit. Series, EPA
670/4-73-001.
(ii) Anonymous. 1975. Standard Methods for the Examination
of water and Waste water. 14th Ed. American Public Health Assoc.
Washington, D.C.
§ 154-9 Freshwater aquatic invertebrate acute bioassay; Tier I.
(a) When repaired. (1) Toxicity (or toxic-like effects)
data on a freshwater aquatic invertebrate are required by 40 CFR
§ 158.165 to support the registration of each end-use product in-
tended for outdoor terrestrial application and each manufacturing-
use product that legally may be used to formulate such an end-use
product. See 40 CFR § 158.50 and $ 158.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the forsmlator*' exemption
and who, as -a general rule, is responsible for submission of the
required data.
(2) If the pesticide will be introduced directly into an
aquatic environment when used as directed, then it must be tested
as indicated in $§ 72"1 through -7 of Subdivision E and as required
by 40 CFR | 158.145.
-------
216
(b) Test standards. The general and specific test standards
for biochemical pesticide* are the sane as those set forth in
Subdivision C in §$ 70-3 and 72-1, respectively, with the following
exception: One maximum hazard concentration aay be tested in lieu
of conducting a definitive X£so or 1050 test if exposure of 30
organisms to a concentration of 1/000 time* the expected aquatic
environmental concentration or 100 mg/1 of water (whichever is
higher) produces less than 50 percent Mortality.
(c) Reporting of data* The provisions for reporting of data
are the same as those set forth la Subdivision t of the guidelines
(§ 72-2).
(d) Tier progression. (1) Biochemical agents that meet one
or more of the following three criteria will not require testing at
Tier II, [Environmental Fate ($§ 155-1 through -14)} except as
noted in paragraph (d)(2) below:
(i) The use pattern or formulation precludes the possibility
of significant exposure of aquatic animals when the pesticide is
used as directed; or
(ii) The amount of biochemical applied is small (less than
0*7 ounces (20 grams) active ingredient per acre); or
(ili) The estimated volatility of the biochemical is high
(equal to oz greater than 5x10~5 ata. o3/aol).
(2) Biochemical pesticides that meet one or more of the above
three criteria require testing at Tier II [environmental fate ($f
155-1 through -14)} if any of the following occur:
(i) Signs of abnormal behavior are reported in the Tier I
test at concentrations less than or equal to the maximum expected
concentration in water; or
(ii) The maximum expected concentration in water is equal to
or greater than 0.1 the LC$o determined by testing outlined in this
section; or
(ill) The maximum expected concentration in water is equal to or
greater than 0.01 the LCso determined by testing outlined in this
section/ and adverse effects on growth, development, or reproduction
may be expected, based on Tier X test data, available^environmental
fate data (e-.g., from the product's research and development), use
pattern information, or available effects data on phylogenetically
similar target species.
-------
217
(3) If the criteria in paragraph (d)(1) of thia auction ar«
not act, than additional testing at Tier II (}§ 155-1 through -14)
is required.
(e) References. (1) Examplaa of aceaptabl* protocols for
conducting a freshwater aquatic invertebrate acuta toxicity study
may be found in the following reference. Aquatic invertebrate
teat temperatures found in reference (ii) are acceptable with tha
exception of 17»C for Daphnia »pp. Daphnia should be tested at 20*
* 1*C.
(i) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates, and Amphlobians.
American Society for Tasting and Materials, 1916 Race Street,
Philadelphia, PA 19103.
(ii) Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates and Amphibians. U.S. tovironmental Protec-
tion Agency, Ecol. Res. Series, EPA 660/3-75-009. 61 pp.
£ 2) The following may contain useful background information
for developing acceptable protocols:
(i) Weber, C.E. (ed.). 1973. Biological- field and laboratory
methods for measuring the quality of surface waters and effluents.
U.S. Environmental Protection Agency, Environ. Monit. Series, EPA
670/4-73-001.
(ii) Anonymous. 1975. Standard Methods for the Examination
of Water and Wastewater. 14th Ed. American Public Health Assoc.,
Washington, D.C.
§ 154-10 Plant studies; Tier I.
(a) When required. (1) Data on the toxic effects of a bio-
chemical pesticide on plant growth and development are required as
specified by 40 CFR § 158.165 to support the registration of
each end-use product intended for outdoor application and each
nanufacturing-use product that legally may be used to formulate
such an end-use product* [See f 120-1(e) of subdivision J.] See
40 CFR § 158.50 and $ 158.165 to determine whether these data must
be submitted; Section II-B of this subdivision contains an addi-
tional dicussion of the femulators' exemption and who, as a gen-
eral rule, is responsible for submission of the required data.
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218
(2) When plant studies arc required as outlined in paragraph
(a)(1) of the section, the indicated teats (in Subdivision J) should
be conducted for the following use patterns:
(i) Plant-controlling biochemical pesticides* The target
area phytotoxicity teat ($121-1), the seed germination/seeding
emergence and vegetative vigor tests ($ 122-1) and the growth and
reproduction of aquatic plant tests (} 122-2) should be performed.
(ii) Animal-controlling biochemical pesticides* The seed
germination/seedling emergence and vegetative vigor tests (f 122-2)
should be performed except where:
(A) The material is to be used in a contained manner rather
than in a general broadcast or band manner;
(B) The material is to be used as a broadcast treatment at
levels less than three times the naturally-occurring level; or
(C) The material occurs naturally in the plant community type
where usage of the product is intended.
(b) Test standards* The pbytotoxicity studies as outlined in
this subdivision should meet the Subdivision J general test standards
(§ 120-3) and specific test standards [§ 121-Kb), 122-1 (b), and
122-2(b)] for the appropriate tests with the following exceptions.
(1) Test substance. A typical end-use product shall be
tested.
(2) Dose levels. One concentration level equal to not less
than the •>»*< «^«« label rate should be tested where the active
ingredient application solution concentration is 10 ppb or greater.
The phrase "the maximum label rate" means the amount of active
ingredient that may be used per land area or applied directly to
the surface of a 15-cm or 6-inch column of water.
(c) Reporting. In addition to the general information required
in $ 120-4(b) of Subdivision J, the reporting requirements for the
other tests [§§ 121-1(c), 122-Kc), and 122-2{c)] should be followed.
(d) Tier progression. (1) If an adverse Effect or response
on plant growth and development for any terrestrial plant species
is 25 percent or greater with respect to the control or 50 percent
or greater for aquatic plants, then testing at tier*il tEnvironmental
Fate, H t55-1 through 155-14) is required as specified in 40 CTR
§ 156.165.
(2) If less than a 25 percent adverse effect or response is
noted for terrestrial plants or 50 percent for aquatic plants no
additonal testing at higher tiers is ordinarily necessary. The
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.219
Agency, however, After reviewing the data, may require certain
additional tests to determine a more accurate no observed effect
level.
§ 154-11 Hontarget insect teating; Tier I.
(a) When required. (1) General. Tier I testing for effects
of biochemical pesticides on nontarget insects is required on a case-
by-case basis as specified by 40 CFR 4 158.165 to support the regis-
tration of each end-use product, and of each manufacturing-use
product that legally may be used to formulate such an end-use
producta
(2) Tests required, (i) Terrestrial insects. The registrant
oust report any adverse effects noted during efficacy testing (J
156-2), and/or any data that indicate potential for adverse effects
on nontarget insects.
(ii) Aquatic insects. (A) If the biochemical is introduced
directly into an aquatic environment when used as directed, testing
oust be conducted in accordance with Subdivisions E and L.
(B) If the biochemical is not introduced directly into an
aquatic environment when used as directed, testing must be conducted
as specified in § 154-9.
(b) Test standards. Studies conducted in accordance with
paragraph (a)(2)(ii)(A) should meet the applicable requirements
outlined in §140-3 of Subdivision L. Studies conducted in accordance
with paragraph (a)(2) (ii)(B) should meet the applicable requirements
outlined in §| 150-3 and 154-9 of this subdivision.
(c) Reporting and evaluation of data. The test report should
contain the information required in Subdivision L (§ 140-4 and
other applicable sections) or this subdivision (§ 150-4 and
other applicable sections), as appropriate.
(d) Tier progression. (1) Terrestrial insects. Tier II
testing ($ series 155) is required by 40 CFR $ 158.165 according to
the following criteria:
(i) If adverse effects are noted during efficacy testing ($
156-2), or
(ii) If other data exist which indicated potential for adverse
effects on nontarget terrestrial insects.
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220
(2) If neither of the criteria in paragraph (d)(1)(i) or (ii)
of this section apply, no further tasting is necessary.
(3) Aquatic insects, (i) If the biochemical is introduced
directly into an aquatic environment when used as directed, testing
is required as outlined in Subdivisions E and L.
(ii) If the biochemical is not introduced directly into an
aquatic environment when used as directed, testing is required as
specified in § 154-9.
Group X-2: Tier III testing.
§ 154-12 Terrestrial wildlife testing; Tier III.
(a) When required. Data on the effects of a biochemical
pest control agent on terrestrial wildlife are required by 40 CFR
§ 158.165 as outlined in section series 71 of Subdivision E 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, if:
(1) Environmental fate characteristics indicate that the
estimated concentration of the biochemical pesticide in the
terrestrial environment is equal to or greater than 1/5 the avian
dietary LC50 or the avian single dose oral LD50 (converted to ppm) ;
or
(2) The pesticide or any of its metabolites or degradation
products are stable in the environment to the extent that potentially
toxic amounts may persist in the avian feed.
(3) See 40 CFR $ 156.50 and $ 156.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the femulators' exemption
and who, as a general rule, is responsible for submission of the
required data.
(b) Test standards. The test standards in $ 70-3 and ${ 71-2
through -5. of Subdivision E apply.
(c) Reporting and evaluation of data. The reporting and
evaluation provisions in J 70-4 and §§ 71-2 through -5 of Subdivi-
sion E apply.
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'221
(d) Tier'progression. rurth«r testing -shall be conducted «•
specified in f§ 71-2 through -5 of Subdivision E.
154-13 Aquatic animal testing; Tier III.
(a) When required. Data on the 40 CTR $ 158.165 effects of a
biochemical pest control agent on aquatic animals are required to
support the registration of each end-use product intended for out-
door application and each manufacturing-use product, that legally
•ay be used to £ormulate auch an end-use product if environmental
fat* characteristic* indicate that the estimated environmental
concentration of the biochemical agent in the aquatic environ-
ment is equal to or greater than 0*01 of any KCSO or LCSO deter-
mined in testing outlined by § 154-8 or -9. See 40 CTR $ 158.50
and $ 158.165 to determine whether these data must be submitted;
Section ZZ-B of this subdivision contains an additional discus-
sion of the f emulators' exemption and who/ as a general rule, is
responsible for submission of the required data.
(b) Test standards, the test standards in $ 70-3 and §§ 72-1
through -7 of Subdivision C apply.
(c) Reporting and evaluation of data. Wie reporting and
evaluation provisions in § 70-4 and $§ 72-1 through -5 of
Subdivision E spply.
(d) Tier progression. Further testing shall be conducted as
outlined in H 72-1 through -7 of Subdivision E and required by 40
CFR § 158.165.
154-14 Plant studies; Tier III.
(a) When required. Data on the effects of a biochemical pest
control agent on plant growth and development are required by 40 CTR.
f 158.165 on a case-by-case basis to support the registration of
each end-use product agent intended for outdoor application and
each manufacturing-use product that legally may he «s*d to formulate
such a product where the material may be moved from the site of
application by air, soil, or water. The extent of movement will
be determined by the Tier ZZ environmental fate tests (see $ series
155). [See { 120-1(e) of Subdivision J.) Refer 40 CTR § 158.50
and § 158.165 to determine whether these data must be submitted;
Section ZI-B of this subdivision contains an additional discussion
of the formulators' exemption and who, as a general rule, is
responsible for submission of the required data.
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222
(b) Test standard*. The test standard* in Tiers II through
III <$ 123-1 through § 124-2) of Subdivision J apply.
(c) Reporting. -The reporting provisions in Tiers II through
III (\ 123-1 through $ 124-2] of Subdivision J apply.
(d) Tier progression. The tier progression criteria in Tiers
II through III [§ 123-1 through f 124-2] of Subdivision J apply.
154-15 Nentarget insect testing; Tier III.
(a) When required. Data on the effects of a biochemical pest
control agent on nontarget insects are required by 40 CTR § 158.165
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/ when results of
Tier I tests ($ 154-11) indicate potential adverse effects on
nontarget insects and results of Tier XI tests (section series
155) indicate exposure of nontarget insects. Tier III testing
should only be performed following consultation with the Agency.
(b) Test standards. The test standards in § 140-3 of
Subdivision L apply.
(c) Reporting and evaluation of data. The reporting and
evaluation requirements in $ 140-4 of Subdivision L apply.
(d) Tier progression. None.
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223
Subserie* 154B: MXCROBXAL AGENTS
Group B-1: Ti«r I Testing.
§ 154-16 Avian single-dose oral toxicity «nd pathogenicity tests
Tier I.
(•) When required. Data on the avian acute oral toxicity of *
microbiel pest control agent are required by 40 CTR $ 1SB.165 to
support the registration of each end-use product intended for out-
door application and each aanufacturinguse product that legally nay
be used to formulate such an end-use product. See 40 CFR § 158.50
and § 158.165 to determine whether these data oust be submitted;
Section II-B of this subdivision contains an additional discussion
of the formulators' exemption and who, as a general rule, is
responsible for submission of the required data.
(b) Test standards. Data should be derived from tests that
satisfy the general test standards in § 150-3.of this subdivision
and the following test standards:
(1) Test substance. The technical grade of each active ingre-
dient in the product shall be tested.
(2) Speciea. Testing should be performed on one avian species
(preferably bobwhite quail or mallard duck).
(3) Age. Birds used in this test should be from 10 to 17 days
old at the beginning of the test period. Within a given test, all
birds should be of the same age.
(4) Controls, (i) A negative control group is necessary;
(ii) A concurrent control group is recoomended and should be
treated with the technical grade of the active ingredient containing
inactivated microbial agent.
(5) Number of birds per dosage level* Each treatment and
control should contain at least 10 birds. When only one treatment
group is tested, at least 30 birds should be tested at that level.
(6) Maximum hazard dosage level. The highest dosage level
tested should not be less than 10 times the adjusted host equivalent
amount. If the 10x amount is not feasible, a 5x or 2x amount should
be tested. A reason shall be provided to support any reduction in
the highest dosage level from 10 times the adjusted host equivalent
amount.
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224
(7) Determination of an LDSO. Th« test data must establish
that the avian single-dose oral LDSO or ED50 la greater than the
maximum hazard dosage level, or oust establish an LDSO or ED50
valut and corresponding 95 percent confidence intervals, if possible.
(6) Duration of test. Control and treated groups should be
observed for at least 30 days after dosing.
(c) Reporting and evaluation of data. In addition to the
information specified in § 150-4 of this subdivision/ the test
report should contain the following information:
(1) Age of the birds tasted;
(2) Mean body weights for each test and control group at
weekly intervals;
(3) Diet used;
(4) Pen dimensions;
(5) Ambient temperature and humidity; (6) Photoperiod and
lighting;
(7) Total feed consumption for each test and control
group at weekly intervals»
(8) Preparation of test material;
(9) Amount of test material dosed per bird;
(10) Amount of vehicle dosed per bird, if a vehicle is used;
(11) Number of birds per treatment level;
(12) Number of controls used;
(13) LDSO or ED50 in mg/fcg, with 95 percent confidence limits,
if obtained;
(14) Methods used for calculation of LDSO;
(15) Slope of the dose response line, if obtained;
(16) Time and date of sortalities;
(17) All signs of intoxication, abnormal behavior,
and regurgitation (if any occurs);
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225
(18) Reports of any pathogenic effect* or pathological changes;
(19) Results of gross necropsies conducted on enough birds to
characterize any gross lesions; and
(20) Reasons to support a reduction (if any) in the highest
dosage level required by paragraph (b)(6) of this section.
(d) Tier progression. (1) If any toxic or pathogenic
effects axe observed at the maximum hazard dosage level in this
study, testing at Tier II, environmental expression testing
(§§ 155-15 through -20), is required as specified by 40 CFR
i 158.165.
(2) If toxic or pathogenic effects are not observed in this
study, additional testing at higher tiers is ordinarily not
necessary.
(e) References. The following references are provided for use
in the development of acceptable test protocols for conducting an
avian single dose oral toxicity test with a microbial pest control
agent:
(1) Xgnoffo, C.M. 1973. Effects of entomopathegens on verte-
brates. Annals K.Y. Acad. Sei. 217:141-164.
(2) 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.
(3) Friend, M., and D.O. Trainer. 1971. Experimental duck
virus hepatitis in the mallard. Avian Disease 16(4): 692-699.
(4) Narayanan, JC., G. Santharam, S. Easwaramoorthy, and S.
Jayaraj. 1976. Lack of susceptibility of poultry birds to nuclear
polyhedrosis virus of groundnut red-hairy caterpillar, Amsacta
albistriga (W.). Indian J. Exper. Biol. 16(12):1322-1324.
(5) Podgwaite, J.D., and R.R. Galipeau. 1978. Effects of
nucleopolyhedrosis virus on two avian predators of the gypsy moth.
USDA, For. Serv. Res. Hote, HE - 251, 2 pp.
(6) Summers, M., R. Engler, X..A. Falcon, and 1.. Vail, eds.
1975. Guidelines for Safety Testing of Baculoviruses, Pp. 179-184
in Baculoviruses for Insect Pest Control. Safety Considerations.
American Society for Microbiology, Washington, D.C.
(7) Tucker, R.K., and M.A. Haegele. 1971. Comparative acute
oral toxicity of pesticides to six species of birds. Toxicol.
Appl. Pharmacol. 10:57-65 (see pp. 57-59).
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.226
(8) Wolf, K. 1975. Evaluation of the exposure of fish and
wildlife to nuclear polyhedrosis and granules is viruses. Pp. 109-111
in Baculoviruses for Insect Past Control. Safety Considerations.
American Society for Microbiology, Washington, D.C.,
154-17 Avian injection pathogenicity tests Tier I.
(a) Whan required. Data OB the aviaa acute injection patho-
genicity of a aicrobial pest control agent are required by 40 CTR
§ 158.165 to support the registration of each end-use product in-
tended for outdoor application and each manufacturing-use product
that legally may be used to formulate such an end-use product.
See 40 CPU $ 158.50 and § 158.165 to determine whether these data
must be submitted; Section ZI-B of this subdivision contains an
additional discussion of the formula tors' exemption and who, as a
general rule, is responsible for submission of the required data.
(b) Test standards. Data sufficient to satisfy the require-
ment in paragraph (a) of this section should be derived from tests
which satisfy the purposes of the general test standards in
§ 150-3 of this subdivision, and all the following test standards:
( 1 } Test substance. Data should be derived from testing
conducted with the technical grade of each active ingredient in the
product .
(2) Species . Testing should be performed on one avian species
(preferably bobwhite quail or mallard duck).
(3) Age. Birds used in this test should be from 10 to 17
days old at the beginning of the testing period. Within a given
test, all birds shall be the sane age.
(4) Controls . (i) A negative control group is necessary;
(ii) A concurrent control group is necessary and should be
treated with the technical grade of the active ingredient containing
inactivated microhial agent.
(iii) Two untreated contact control birds are recommended,
and should be placed in with the treatment group receiving the
hacard dosage*
(5) Number of birds per dosage level. Each treatment and
control group should contain at least 10 birds. When there is only
one treatment group at least 30 birds should be tested at that
treatment level.
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227
<6) Route of exposure. The test arterial should be adminis-
tered by intraperitoneal or intravenous injection. The intravenous
route may not be feasible because of problems related to the size
of particles in the inoculum or technical problems related to the
age or sice of the birds.
(7) Maximum hatard dosage level. The highest dosage level
tested should not be less than an. amount equal to one adjusted host
equivalent. If this amount is not feasible, a 1/2x, 1/5x or 1/10x
amount should be tested. A reason shall be provided to support any
reduction in the highest dosage level from an amount equal to the
adjusted host equivalent amount.
(8) Determination of an EDSO. The test data must establish
that the avian injection XD50 is greater than the maximum hazard
dosage level, or sequentially lower levels shall be tested to
provide a dose-response series which includes at least one level in
which no mortality occurs. If possible, an ZD50 value and corre-
sponding 95% confidence intervals shall be established.
(9) Duration of test. Control and treated birds should be
observed for at least 30 days after dosing.
(c) Reporting and evaluation of data. In addition to the
information specified in J 150-4 of this subdivision, the test
report should contain the following information:
(1) Age of the birds tested;
(2) Mean body weights for each test and control group at weekly
intervals;
(3) Diet used;
(4) Pen dimensions;
(5) Ambient temperature and humidity;
(6) Photoperiod and lighting;
(7) Total feed consumption for each teat and control group at
weekly intervals;
(8) Preparation of test material;
(9) Amount of test material injected per bird;
(10) Amount of vehicle injected per bird, if vehicle is used;
(11) Number of birds per treatment level;
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228
(12) Number of controls used;
<13) Time and date of mortalities;
(14) O50 in mg/kg/ with 95% confidence limit*, if obtained;
(15) Results of grot* necropsy conducted on all birds dying
before termination of the test and on a representative sample of
those that survived, and on the two contact control birds. The
necropsy report should include any evidence of multiplication of
microbes (e.g. lesions), att
(i) The site of injection! and
(ii) Distant sites including liver, kidney, lungs, spleen,
cerebrospinal system, gastrointestinal system, and circulatory
system)
(16) A description of the methods used to assess the cause
and effects of any lesions noted where there is evidence that the
mierobial agent is multiplying in the bird;
(17) Assessment of any effects noted; and
(18) Reason to support reduction in highest dosage section.
(d) Tier progression. (1) If any pathogenic effects are
observed at the maximum hazard dosage level in this study, testing
at Tier II [environmental expression testing (§§ 155-15 through
-20)] is required as specified in 40 CFR $ 158.165.
(2) If no pathogenic effects are observed in this study, no
additional testing at higher tiers ordinarily is necessary.
(e) References. The following references are provided for
use in the development of acceptable test protocols for conducting
an avian injection pathogenicity test with mierobial pest control
agents:
(1) Ahmed, M.M., and D.H. Walker. 1979. The metabolism of
DDT in vivo by the Japanese quail (Coturnix eoturnix japoniea).
Pesticide Bioehea. Physiel. 10:40-48.
(2) Friend, M., and D. 0. Trainer. 1974a. experimental DDT-
Duek hepatitis virus interaction studies. J. tfildl. Manage.
33(4) :387-895.
(3) . 1974b. Experimental Dieldrin-Duck hepatitis
virus interaction studies. J. Wildl. Menage. 38(4):896-902.
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229
(4) .. . 1972. Duck hepatitis virus interaction with
DOT and Dieldrin in adult mallards. Bull. Environ. Contam. Toxicel.
7(4):202-206.
(5) Friend, M., and D.O. Trainer. 1971. Experimental duck
virus hepatitis in the mallard. Avian Disease 16(4):692-699.
.(6) . . 1970. Polychlorinated biphenyl: interaction
with duck hepatitis virus. Science 170(3964):1314-1316.
(7) Sinners, M., R. Engler, X..A. Falcon/ and p. Vail/ eds.
1975. Pp. 179-184 in Guidelines for Safety Testing of Baculoviruses.
Baculcviruses for Insect Pest Control: Safety Considerations.
American Society for Microbiology, Washington, D.C.
§ 154-18 wild mammal toxieity and pathogenieity testing; Tier I.
(a) When required. Data on wild mammal toxieity and patho-
genieity may be required by 40 CTR § 158.165 on a case-by-case basis
to support the registration of end-use products intended for outdoor
application and manufacturing-use products that legally may be used
to formulate such end-use products. The toxieity and pathogenieity
data outlined in Subdivision F for evaluating hazard to humans and
domestic animals are normally adequate to indicate 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 microbial pest control agent. An example of
one circumstance when such data may be required is the situation
in which data indicate that there is considerable variation in
sensitivity of different mammalian species to the effects of a
microbial pest control agent, and there is evidence that wild
mammals are heavily exposed to microbial pest control agents.
See 40 CTR § 158.50 and §158.165 to determine whether these data
must be submitted; Section IZ-B of this subdivision contains an
additional discussion of the formula tors' exemption and who, as a
general rule, is responsible for submission of the required data.
(b) Test standards. Data should be derived from tests that
satisfy the general test standards in | 150-3 of this subdivision
and the following test standards:
(1) Test substance. Data shall be derived from testing con-
ducted with the technical grade of each active ingredient in the
product.
(2) Species. Testing should be performed on a mammalian spe-
cies representative or indicative of those found in the area(s)
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230
likely to be affected by the proposed use pattern(s). Test animals
may be reared in pens or captured In the wild, and should be pheno-
typically indistinguishable from wild mammals. Endangered or
threatened animal* shall not be used.
(3) Controls* (1) A negative control group is necessary; and
(ii) X concurrent control group is necessary and should be
treated, when possible, with the technical grade of the active
ingredient containing inactivated (e.g., autoclaved) microbial
agent.
(4) Route of exposure. The test material"should be admi-
nistered by gavage (acute oral dose) or by injection (iatraperiton-
al or intravenous), the route shall be determined after consulta-
tion with the Agency.
(5) Maximum hazard dosage level, the standards for maximum
hazard .dosage level/ determination of an BD50, and duration of
test that are found in the avian single dose oral toxicity and
pathogenicity test $ 154-16(b) and the avian injection patho-
genicity test § 154-17(b) apply also to the respective tests on
(c) Reporting and evaluation of data. In addition to the
information specified in J 150-4 of this subdivision, test reports
should contain the sane information required for the avian single-
dose oral toxicity and pathogenieity test $ 154-16(c) and the avian
injection pathogenicity test $ 154-17(0), adapted appropriately for
mammalian test procedures!
(d) Tier progression. (1) If any toxic or pathogenic effects
on mammalian species are observed at the ff^iimjr1 hazard dosage level
in this study, testing at Tier II [environmental expression testing
(B 155-15 through -20)] is required as specified by 40 CFft § 154.165.
(2) If toxic or pathogenic effects are not observed in this
study, additional testing at higher tiers is ordinarily not necessary.
(e) References. The following references are provided for
use in the development of acceptable test protocols for conducting
wild maaswl toxicity and pathogenicity tests with microbial pest
control agents:
(1) Agr. Res. Service, BSDA Animal Disease and Parasite
Research Division. 1969. The toxicity of some organic herbicides
to cattle, sheep, and chickens. A.R.S. Production Research Report
No. 106.
(2) Barnes, R.W., C.F. Hsincke, W.C. McLane, and C.S. Rehn-
borg. 1970. Long-tera feeding and other toxicitypathogenicity
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231
studies on rets using a coaBaarciel preparation of the nuclear-
polyhedrosis virus of Heliothis zee. J. Invert. Pathol. 16:112-
115.
(3) Fisher/ R., and L. Rosner. 1959. Toxicology of the
microbial insecticide, Thuricide. Agriie. Food Ch«a. 7{10)t686-688.
(4) Ignoffo, C.M. 1973. Effects of entomopathogens on
vertebrates. Annals N.Y. Acad. Sci. 217:141-164.
(5) Ignoffo, C.M. 1971. Xntraperitoneal injection of white
mice with nucleopolyhedrosis virus of the beet arayworm/ Spodoptera
exigua. J. Invert. Pathol. 17(3)i453-454.
(6) Ignoffo, C.M., W.K. Barker, and C.W. McCoy. 1973. Lack
of per os toxicity or pathogenieity in rats fed the fungus. Hirsute11*
thompsonii. Zntomephaga 18(3):333-335.
(7} Ignoffo, C.M., C. Garcia, R.W. Kapp, and w.B. Coate.
1975. An evaluation of the risks to manuals of the use of an ento-
oopathogenic fungus, Somiraea rileyi, as a microbial insecticide.
In; Baculoviruses for Insect Pest Control: Safety Considerations.
selected papers from EPA/USDA Working SynposiuB, Amer. Soc.
Microbiology, Washington, D.C.
(8) Ignoffo, C.M., and A.M. Heiopel. 1965. The nuclear poly-
hedrosis virus of Heliothis zea (Boddie) and Heliothis vireseens
(Fabricus) Part V. Toxicity-pathogenicity of virus to white mice
and guinea pigs. J. Invert. Pathol. 7:329-340.
(9) Ignoffo, C.M./ J.J. Petersen, B.C. Chapman, and J.F.
Novotny. 1974. Lack of susceptibility of mice and rats to the
mosquito nematode, Reesimermis nielseni, Tsai and Grundmann. Mos-
eruito News 34(4) :425-428.
(10) Lamanna, C., and L. Jones. 1963. Lethality for mice of
vegetative and spore forms of Bacillus cereus and Bacillus eereus-
like insect pathogens injected intzaperitoneally and subcutaneously.
J. Bacteriology 85:532-535.
(11) Lautenschlager, R.A., C.H. JCircher, and J.D. Podgwaite.
1977. Effect of nucleopolyhedrosis virus on selected mannalian
predators of the gypsy Both. USDA, For* Serv. Ass* Paper, NC-377,
6p.
(12) Lauterjchlager, R.A., and J.D. Podgwaite. 1979. Passage
of nucleopolyhedrosis virus by avian and aaonalian predators of the
gypsy moth, Lymantria dispar. Environ. Entomol. 8(21:210-214.
(13) Lautenschlager, R.A., and J.D. Podgwaite. 1977. Passage
of infectious nuclear-polyhedrosis virus through the alimentary
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232
tracts of two small mammal predators of the gypsy moth, Lymantria
dispar. Environ. Entonol. 6(5):737-738.
(14) Meinacke, C.F., W.C. McLane, and C.S. Rehnborg. 1970.
Toxicitypathogeaicity studies of a nuelear-polyhedrosis virus of
Heliothis tea in white mice. J. Invert. Pathol. 15:10-14.
(15) Pounds, J.G. 1977. Safety and potential hazard* of the
entcmopathogen Mattesia troqedermae to non-target species. Ph.D.
Thesis, University of Wisconsin, Madison, Wisconsin. 131 pp.
(16) Summers, M., R. Eagler, L.A. Galcon, and P. Vail, eds.
1975. Pp. 179-184 in Guidelines for Safety Testing of Baculoviruses
for Insect Pest Control: Safety Considerations. American Society
for Microbiology, Washington, O.C.
(17) Smirnoff, W.A., and C.F. MacLeod. 1964. Apparent lack
of effects of orally introduced polyhedrosis virus on mice and of
pathogenicity of rodent-passed virus for insects. J. Insect Pathol.
6:537-538.
(18) watts, D.M., R.F. Tamnariello, J.M. Dalrymple, B.F.
Eldridge, P.K. Russell, and F.E. Topi Jr. 1979. Experimental
infection of vertebrates of the Pocomoke Cypress Swamp, Maryland
with Keystone and Jamestown Canyon viruses. Am. J. Trop. Med. Hyg.
28(2):344-350.
(19) Webb, R.E., and H. Borsfall, Jr. 1967. Endrin resistance
in the pine mouse. Science 156:1862.
(20) Wolf, K. 1975. Evaluation of the exposure of fish and
wildlife to nuclear polyhedrosis and granulesi« viruses. Pp. 109-
111 in Baculoviruses for Insect Pest Control: Safety Considerations.
American Society for Microbiology, Washington, D.C.
§ 154-19 Freshwater fish toxieity and pathogenicity testing: Tier I.
(a) When required. Data on pathogenicity and/or toxieity to
fish are required by 40 CTR § 158.165 to support the registration of
each end-use product intended for outdoor applicSreSMrand each
manufacturing-use product that legally Bay be used to formulate
such an end-use product.
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233
(b) Test standards. Data should b€ derived from tests that
satisfy the general t«st standards in $ 150-3 of this subdivision,
and the following test standards:
(1) Test substance, (i) Data to support the registration of
an and-use formulated product or a manufacturinguse product shall
be derived from tasting conducted with:
(A) The technical grade of each active ingredient in the
product when the test substance is added directly to the test water;
and
(B) The sest challenging form (in terms of pathogenicity and
toxicity) of each active ingredient (microorganism) in the product
when the test substance is administered in the diet or by injection.
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234
(v) Fish should weigh between 0.5 and 5.0 grams and b« from
the MB* year class, the standard length of the Ionise fish should
be no nor* than twice that of th* shortest fish.
(3) Method of pesticide administration, (i) The tast
substance should be administered as a suspension directly into the
water (i.e./ aqueous exposure).
(ii) Two additional methods of pesticide administration should
be considered and used in combination with the aqueous exposure in
the same test, whenever possible. The two Aethods are:
(A) Dietary administrations, food to be administered in the
fora of target bast organisms infected with the microbial agent or
feed supplemented with microbial agent; and
(B) Administration by intraperitoneal injection.
(4) Treatment concentrations, (i) If the test substance pro-
duces a toxin, then a sufficient number of treatment concentrations
should be tested to determine toxicity as described in paragraphs
(b)(5)(ii)(A),(B) and (b)(5Miii) of this section.
(ii) If the test substance does not produce a toxin, or no
toxin has been identified, then a single, replicated, maximum hazard
exposure may be tested. Treatment concentrations or doses should be
selected as follows, whenever possible:
(A) At a minimum, the concentration in the test water (for
aqueous exposure) should, whenever possible, equal the maximum
calculated pesticide concentration in a six-inch layer of water
immediately following a direct application to a six-inch layer of
water;
(B) Peed used in the dietary exposure should be supplemented
with the test substance to achieve a microbial concentration greater
than or equal to the host equivalent; and
(C) The injected test substance should contain a concentration
of active ingredient equal to the adjusted host equivalent.
(iii) The fish infertility test(s) conducted in Tier III may
require the use of lower treatment concentrations and/or a greater
number of treatment concentrations in order to *3btikt*£sse the
concentration (or dose) response relationship or the minima
effective concentration.
(5) Determination of toxieity or pathoqenieity. (i) The
pathogenicity of the test substance on the test organisms following
a sufficiently long period of exposure and observation should be
determined.
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235
(ii) If the test substance produces a toxin, then the following
shall be determined:
(A) A precise LCSO value with 95 percent confidence intervals;
or
(B) That the LCs0 is greater than 100 mg/1 or 1,000 times the
estimated environmental concentration, whichever is higher.
(iii) If data are submitted to satisfy either criterion in
paragraph (b)(5)(ii)(B) of this section, the data should be derived
from a study containing at least 30 organisms tested at concentra-
tions equal to or greater than the applicable criterion (100 mg/1
or 1,000x estimated environmental concentration).
(e) Reporting and evaluation of data. In addition to
information meeting the general reporting requirements of $ 150-3
of this subdivision, a report of the results of a fish toxicity and
infactivity test would include the following:
(1) LC5Q data (if the test substance produces a toxin).
(i) Such data should show:
(A) The 96-hour LCso/ the corresponding 95 percent confi-
dence intervals, and, when possible, the LCso values at 24 hour
intervals for the duration of the test; or
(B) That the LC50 is greater than 1,000 times the expected
environmental concentration or 100 mg/1.
(ii) If the data submitted in accordance with paragraph
(c)(1)(i)(B) of this section indicate that the LCso ia Sweater than
1,000 tines the expected environmental concentration of the pesticide,
then the basis for calculating the estimated environmental concen-
tration should be shown.
(2) Detailed description of the steps taken to determine
microorganism dissemination, replication, or survival in the test
animals tissues, organs, or fluids:
(3) Detailed description of dilution water, including:
(i) Source;
(ii) Chemical characteristics (e.g., dissolved oxygen content, pH,
dissolved salts); and
(iii) Pretreatment (if any);
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236
(4) Detailed description of the test. Including:
(i) Design;
(ii) Containers;
(iii) Medium (e.g., depth and volume);
(iv) Treatments;
(y) Method of exposing fish to the test substance (e.g.,
placing microbial agent in water which already contains fish or
placing fish in water which already contains the" mierobial agent)>
(vi) Number of organisms per treatment;
(vii) Loading (weight of organisms per unit volume of medium
or unit surface);
(viii) Lighting, acclimation., and test temperatures (averages
and range);
(ix) Amount of test substance administered by each route of
exposure; and
(x) Any unusual feature of the test method;
(5) Detailed descriptions of methods (or references to
established methods) used for all chemical analyses of water for
chemical content and toxicant concentrations;
(6) Detailed description of methods used for all mierobial
analyses of water and test organisms, and results of such analyses,
including validation studies;
(7) Detailed description of the effects of exposure to the
test substance including:
(i) The criteria used to determine the effects;
(ii) Percentages of organisms that died or showed effects of
treatment; and
(iii) A summary of these observations; and
(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 XI (environmental expression
testing (§§ 155-15 through -20}] is required as specified by 40 CFR
158.165.
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237
(.2) Further tier tea ting is not necessary if results of this
study do not indicate -toxic or pathogenic effects.
(e) References. The following may contain useful background
information for developing acceptable protocols:
(1) Anonymous. 1975* Standard Methods for Examination of
Water and Wastewater. 14th Xd. American Public Health Assoc.,
Washington, D.C. 1193 pp.
(2) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Pishes, Macroinvertabrates, and Amphibians.
American Society for Testing and Materials, 1916 Race Street/
Philadelphia, 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, SPA 660/3-75-009. 61 pp.
(4) Hetrick, P.M., M.D. Knittel, 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, Mew York.
(6) Ignoffo, C.M. et al. 1973. Susceptibility of aquatic
vertebrates and invertebrates to the infective stage of the mosquito
nematode Reesinermis nielseni. Mosquito News 33(4):599-€02.
(7) Macek, K.J., S.R. Petrocelli, and B.H. Sleight III.
1979. Considerations in assessing the potential for, and significance
of, biomagnification of chemical residues in aquatic food chains.
ASTM STP, pp.251-268, American Society for Testing and Materials,
Philadelphia.
(8) Masxone/ H.M., and G.H. Tignor. 1976* Insect viruses:
serological relationships. Adv. in Virus Res. 20:237-270.
(9) Pagano, J.S., and E. Huang, 1974. The application of TOA-
DNA cytohybridixation to viral diagnostics. In: Viral lamunodiagnosis.
E. Xurstax and R. Morisset, eds. Academic Press, Inc.
(10} Pound, J.F. 1977. Safety and potential hazards of the
entomopathogen Mattesia tregodermae to nontarget species. Ph.D.
Dissertation. 0. Wisconsin.
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238
(11) Reynolds, G.J. 1978. Enzyme labelled Antibody in
histopatholoqy. Qualityline (Winter 1978/1979)»2-10.
(12) Smith, C.E. and M.D. Summers. 1978. Analysis of
baculovirus genomes with restriction endonucleases. Virology 89:517-
527.
(13) 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.
(14) Undeen, A.H., and J.V. Maddox. 1973. The infection of
nonmosquito hosts by injection with spores of the microsporidan
Nosema algerae. J. Invert. Path. 22:258-265.
(15) Van Essen, P.W., and D.W. Anthony. 1976. Susceptibility
of nontarget organisms to Hosema alqerae (Microsporida: Nosematidae),
a parasite of mosquitoes. J. Invert. Path. 28i77-85.
(16) 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.
§ 154-20 Freshwater aquatic invertebrate toxicity and pathogenieity
testing; Tier I.
(a) When required. Data on pathogenicity or toxicity (or
both, when applicable) to an aquatic invertebrate are required by
40 CFR i 158.165 to support the registration of each end-use product
intended for outdoor application and each manufacturing-use prod-
duct that legally may be used to formulate such an end-use product.
See 40 CFR § 158.50 and § 158.165 to determine whether these data
must be submitted; Section II-B of this subdivision contains an
additional discussion of the formulators1 exemption and who, as a
general rule, is responsible for submission of the required data.
(b) Test standards. Data should be derived from tests that
satisfy the general test standards in § 150-3 of this subdivision
and the following test standards:
(1) Test substance, (i) Data to support the registration of
end-use products and manufacturing-use products shall be derived
from tests conducted with:
(A) The technical grade of each active ingredient in the
product when the test substance is added directly to the test water
(aqueous exposure); and
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239
(B) The most challenging fora (in terms of pathogenicity and
toxicity) of each active ingredient (microorganism) in the product,
when the teat substance is administered in the diet or by injection.
(ii) In addition, data from testing with the end-use product
are required to support the registration of any end-use product if
an ingredient in the end-use product other than the active ingredient
is expected to:
(A) Enhance the toxicity or pathogenicity of the active
ingredient;
(B) Enhance the ability of the active ingredient (microorga-
nism) to survive or replicate in an aquatic environment! or
(C) Independently cause toxicity to aquatic organisms.
(2) Test organisms, (i) Testing should be performed on one
or two aquatic invertebrate species, depending upon the site of
pesticide application as follows:
(A) Terrestrial application: test one species.
(B) Direct application into a freshwater environment: test
two species.
(C) Direct application into an estuarine or marine environment:
test one species*
(ii) Testing of additional aquatic invertebrate species may
be required in Tier I as specified in paragraph (d)(3)(i) of this
section.
(iii) Aquatic invertebrate species likely to prey upon or
scavenge the diseased target host organisms should be tested, when
applicable.
(iv) Immature invertebrates should be used whenever possible.
(3) Method of pesticide administration, (i) The test
substance should be administered as a suspension directly into the
water (i.e., aqueous exposure).
(ii) Two additional methods of pesticide administration should
be considered and used in combination with the aqueous exposure in
the accompanying tests, whenever possible. The two methods are:
(A) Dietary administration: feed to be administered in the
form of target host organisms infected with the aicrobial agent or
feed supplemented with microbial agent; and
-------
240
(8) Administration by injection.
(4) Treatment concentrations. (i) if the test substance
produces a toxin, then a sufficient number of treatment concentra-
tions must be tested to permit a determination of toxicity as
described in paragraphs (b)(S)(ii)(A), (B), and (b)(5)(iii) of
this section.
(ii) If the test substance does not produce a toxin/ or no
toxin has been identified, then a single, replicated, "'rl"^"1 hazard
exposure may be tested. Treatment concentrations or doses shall be
selected as follows:
(A) At a ainjmum, the concentration in the test water (for
aqueous exposure) should, whenever possible, equal the maximum
calculated pesticide concentration in a six-inch layer of water,
immediately following a direct application to a six-inch layer of
water;
(B) Feed used in the dietary exposure should be supplemented
with the test substance to achieve a microbial concentration greater
than or equal to the host equivalent, whenever possible.
(C) The injected test substance should contain, whenever
possible, a concentration of active ingredient equal to the adjusted
host equivalent.
(5) Determination of toxicity or pathogen!city, (i) Satis-
factory data should establish whether the test substance had a
pathogenic or toxic effect on the test organisms during a suffi-
ciently long period of exposure and observation.
(ii) If the test substance produces a toxin, then satisfactory
data must establish either:
(A) A precise tC$Q or LCso value with 95 percent confidence
intervals; or
(B) That the EC$Q or LCso i« greater than 100 mg/1 or 100,000
times the estimated environmental concentration, whichever is
higher.
(ill) If data are submitted to satisfy either criterion in
paragraph (b)<5)(ii)(B) of this section, the data should be derived
from a study containing at least 30 organisms tasted at concentra-
tions equal to or greater than the applicable criterion (100 mg/1
or 1,000 times the estimated environmental concentration).
(c) Peoorting and evaluation of data. In addition to infor-
mation general reporting requirenets of § 150-4 of this subdvision,
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241
a report of the results of an aquatic invertebrate toxieity and
infectivity test would Include the following:
(1) £C$o or ^50 dat* <^* £**• test substance produces a
toxin).
(i) Such data should show:
(X) The EC50 or LCsQr the corresponding 95 percent confi-
dence intervals/ and when possible/ the 2050 or LCso values at
24-hour intervals for the duration of the test} or
(B) That the XCSO or LC50 is greater than 1/000 times the
expected environmental concentration or 100 mg/1, whichever is
higher.
(ii) If the data submitted in accordance with paragraph
(c)(1)(i)(B) of this section indicate that the LC50 or BC50 is
greater than 1/000 times the expected environmental concentration
of the pesticide, then the basis for calculating the estimated
environmental concentration should be shown.
(2) 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,
dissolved salts), and pretreataent (if any).
(4) Detailed description of the test, including:
(i) Design;
(ii) Container;
(iii) Medium (e.g., depth and volume);
(iv) Treatments;
(v) Method of exposing organisms to the test substance (e.g.,
placing chemical in water which contains organisms or placing
organisms in water which contains chemical);
(vi) Number of organisms per treatment;
(vii) Lighting, acclimation/ and test temperatures (averages and
range);
(viii) Amount of test substance administered by each route of
exposure; and
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242
(ix) Any unusual feature of the test method.
(5) Detailed descriptions of methods (or references to
established methods) used for chemical analyses of water for chemical
content and toxicant concentrations.
(6) Detailed descriptions of methods used for all microbial
analyses of water and test organisms/ and the results of such
analyses, including validation studies.
(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 suanary 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) It toxic or pathogenic effects
are observed/ then testing at Tier II [environmental expression
(H 155-15 through -20)1 shall be required specified in 40 CFR
$ 158.165.
(2) If no toxic or pathogenic effects are observed/ then no
further testing at higher tiers is ordinarily necessary, 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 susceptibility of 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 ($$ 155-15
through -20) is required.
(4) It toxic or pathogenic effects are observed in tests con-
ducted in accordance with paragraph (d)(3)(i) of this section, then
testing et Tier II [environmental expression (f{ 415-15 through -20)]
is required. It not, then no further tier testing'is necessary.
(e) References. The following references may contain useful
background information for developing acceptable protocols:
-------
.243
(1) Ancnyaous. 1975. standard Methods for Examination of
Wat«r and Wastewater. 14th Ed. American Public Health Aasoc.,
Washington, 0«C. 1193 pp.
(2) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Pi«he«, Macroinvertebrates, and Amphibian*.
American Society for Testing and Materials, 1916 Race Street,
Philadelphia, PA 19103.
(3) Cconittee on Methods for Toxicity Tests with Aquatic
Organisms. Method* for Acute Toxicity Tests with Fish, Macroinver-
tebrates, and Amphibians. U.S. Environmental Protection Agency,
•col. Res. Series, EPA 660/375-009. 61 pp.
(4) Huang, E., and J.S. Pagano. 1977. Nucleic acid hybri-
dization technology and detection of proviral genomes* Chapter
13 in The Atlas of Insect.and Plant Viruses, K. Maraaorosch, ed.
Academic Press, N.Y.
(S) Zgnoffo, C. M. et al. 1973. Susceptibility of aquatic
vertebrates and invertebrates to the infective stage of the mosquito
nematode, Reeslaermls nielseni. Mosquito News 33(4):599-602.
(6) Lightaer/ O.V., R.R. Proctor, A.K. Sparks, J.R. Adams,
and A.M. Beimpel. 1973* Testing Penaeid shrimp for susceptibility
to an insect Nuclear Polyhedrosis virus. Environ. Entomology
2(4)-.611-613.
(7) Mezzone, H.M., and G.H. Tignor. 1976. Insect viruses:
serological relationships. Advances in virus Research 20:237-270.
(8) Pagano, J.S., and E. Huang. 1974. The application of
RNA-DNA cytohybridization to viral diagnostics. In; Viral
IflBunodiagnoais. E. XurstaJc and R. Morisset, eds. Academic Press,
Inc. New York.
(9) Pound, J.G. 1977. Safety and potential hazards of the
entomopathogen Mattesia trogodermae to nontarget species. Ph.D.
Dissertation. 0. Wisconsin.
(10) Reynolds, C.J. 1978. Kncyne labelled antibody in
histopathology. Qualityline (Winter 1978/1979):2-10.
(11) Smith, C.E. and M.D. Svoners. 1978. Analysis of baculo-
virus genomes with restriction endonucleases. Virology 89:517-527.
(12) Suamers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
1975. Baculoviruses for Insect Pest Control: Safety Considerations.
Selected papers from EPA-OSDA Working Symposium, American Society
for Microbiology, Washington, D.C.
-------
244
(13) Ondeen, A.H., end J.V. Maddox, 1973. The infection of
nomosquito hosts by injection with spores of the microsporidan
Nosema algerae. J. Inv«rt. Path. 22:256-265.
(14> Van Xssen, F.W., and D.W. Anthony. 1976. Susceptibility
of nontarget organism* to Nosema algerae (Microsporida: Nosematidae),
a parasite of mosquitoes. J. Invert. Path. 28:77-85.
(15) Weber, C.E. (ad.) 1973. Biological field laboratory
methods for measuring the quality of surface waters and effluents.
U.S. Environmental Protection Agency, Environ. Monit. Series, EPA-
€70/4-73-001.
154-21 Estuarine and marine animal toxicity and pathogenicity
testsi Tier I.
(a) When required* Data on pathogenicity and/or toxicity to
estuarine and marine •*
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245
ingredient in the end-use formulation other than the active ingredient
J.S expected to:
(A) Enhance the toxicity or pathogenicity of the active
ingredient; or
(B) Enhance the ability of the active ingredient (microorga-
(2) Teat organism, (i) Toxicity and pathogenicity should be
determined for one species of shrimp and one estuarine or marine
fish species.
(ii) Testing of additional estuarine or marine *«j"*i species
may be required in Tier I as specified in paragraph (d)(3)(i) of
this section.
(iii) Bstuarine or marine animals likely to prey upon or
scavenge the diseased target host organisms should be tested, when
applicable .
(iv) Testing of young fish (3 to 6 months old) and immature
invertebrates is preferable. Very young (not yet actively feeding),
spawning, or recently spawned fish should not be tested.
(v) Fish should weigh between 0.5 and 5.0 grams and be from
the same year class. The standard length of the largest fish should
be no more than twice that of the shortest fish.
(3) Method of pesticide administration, (i) The test
substance should be administered as a suspension directly into the
water (i.e., aqueous exposure).
1 ii) Two additional methods of pesticide administration should
be considered and used in combination with the aqueous exposure in
the same test, whenever possible. The two methods are:
(A) Dietary administration: food to be administered in the
form of target host organisms infected with the microbial agent or
feed supplemented with microbial agent; and
(B) Administration by injection.
(4) Treatment concentrations . (i) If the test substance
produces a toxin, then a sufficient number of. treatment concentrations
must be tested to determine toxicity as described in paragraphs
(b)(5)(ii) and (iii) of this section.
(ii) If the test substance does not produce a toxin, or if no
toxin has been identified, then a single, replicated, maximum hazard
-------
246
exposure may be tested. Treatment concentrations or dose should be
selected *» follow:
(A) At a minimum, the concentration in the test water (for
aqueous exposure) should, whenever possible, equal the maximum
calculated pesticide concentration in a 6-inch layer of water
JJBBediately following a direct application to a 6-inch layer of
water.
(B) Feed used in the dietary exposure should be supplemented
with the test substance to achieve a microbial concentration greater
than or equal to the host equivalent, whenever possible*
(C) The injected test substance should contain, if possible/
a concentration of active ingredient equal to the adjusted host
equivalent*
(iii) The estoarine or marine organism toxicity and inf activity
tests conducted in Tier XII may require the use of lower treatment
concentrations and/or a greater number of treatoent concentrations
in order to determine a concentration response relationship or
um «ff«etive concentration.
(5) Determination of toxieity/ or pathogenlcity and infect ivity.
-------
247
(B) That the LC50 or EC50 is greater than 1,000 times the
expected environmental concentration or 100 ag/1, whichever is
higher.
(ii) If the data submitted in accordance with paragraph
(c)(1)(i)(B) of this section indicate that the LC50 or BC50 is
greater than 1,000 tines the expected environmental concentration
of the pesticide, then the basis for calculating the estimated
environaental concentration should be shown.
(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,
dissolved salts), and pretreatment (if any);
(4) Other pertinent details, including:
(i) Design;
(ii) Containers;
(iii) Medium (e.g., depth and volume);
(iv) Treatments;
(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)2
(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
by each route of exposure; and
(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 toxicant concentrations;
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248
(6) Detailed description of methods used in all microbial
analyses of water and test organisms, and the results of such
analyses, including validation studies.
(7) Detailed description of the effects of exposure to the
test substance, including:
(1) The criteria used to determine the effects;
(ii) A statement of the percentage of organisms that died or
showed effects from the treatment; and
(ill) 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
(H 155-15 through -20)] is required as specified in 40 CFR § 158.165.
(2) If no toxic or pathogenic effects are observed, then no
further testing at higher tiers is ordinarily necessary, except as
noted in paragraph (d)(3) of this section.
(3) If efficacy or beneficial insect tests indicate a broad
host spectrum such that susceptibility of estuarlne or marine
invertebrates is indicated, then either:
(i) Additional estuarine or marine invertebrate species must
be tested as described in paragraphs (a) through (c) of this section;
or
(ii) Testing at Tier II [environmental expression (§ 155-15
through -20)] is required.
(4) If toxic or pathogenic effects are observed in tests con-
ducted in accordance with the requirements of this section, then
testing at Tier II [environmental expression (f 155-15 through
-20)1 is required. Otherwise, no further tier testing is necessary.
(e) References. The following may contain useful background
information for developing acceptable protocols:
(1) Anonymous. 1975. Standard Methottl*%or acamination of
Water and Wastewater. 14th Ed. American Public Health Assoc.,
Washington, D.C. 1193 pp.
(2) ASTM Standard C 729-80, Practice for Conducting Static
Acute Toxicity Tests with Larvae of Four Species of Bivalve Molluscs.
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249
American Society for Tasting and Materials, 1916 Race street, Phila-
delphia, PA 19103.
(3) Anonymous. 1978. Biousay Procedures for the Ocean
Disposal Permit Program. D.S. environmental Protection Agency,
Office of Research and Development. EPA-600/9-78-010. 121 pp.
(4) Banner, L.B., C.D. Craft, and D.R. Kimmo. 1975. A salt-
water flow-through bioassay method with controlled temperature and
salinity. Prog. Fish-Cult. 37(3)t126-129.
(5) Clark, J.R., and R. t. Clark, eds. . 1964. Seawatsr
systems for experimental aquariums. D.S. Dept. Int., Pish, and
Wild.Serv., Bur. Sport. Pish. Wild. Res. Rep. Mo. 63, 192 pp.
(6) Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for acute toxicity tests with fish,
macroinvertebratea, and amphibians. U.S. Environmental Protection
Agency, Scol. 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 estuarine
and marine shrimp. Annals M.Y. Aead. Sci. 219:528-536.
(8) 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.
(9) Betricfc, F.M., M.D. Knittel and J.L. Fryer. 1979.
Increased susceptibility of rainbow trout to infectious bematopoetic
necrosis virus after exposure to copper. Appl. Environ. Micro.
37(2):198-201.
(10) Huang, E. and J.S. Pagano. 1977. Hue Hie acid
hybridization technology and detection of proviral genomes. Chapter
13 _in The Atlas of Insect and Plant Viruses. K. Maranorosch, ed.
Academic Press, N.Y.
(11) Ignoffo, C.M., et al. 1973. Susceptibility of aquatic
vertebrates and invertebrates to the infective stage of the mosquito
nematode, Reesinermis nielseni. Mosquito Kew« 33(4) .-599-602.
(12) Lightner, D.V., R.R. Proctor, A.K. Sparks, J.R. Adams,
and A.M. 'Beimpel. 1973. Testing Penaeid shrimp for susceptibility
to an insect Nuclear Polyhedrosis virus. Environ. Entoaol. 2(4)1611-
613.
(13) Macek, K.J., S.R. Petrocelli, and B.B. Sleight III.
1979. Considerations in assessing the potential for, and significance
of bionagnification of chemical residues in aquatic food chains.
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A5TM STP, pp. 251-268, American Society for Testing and Materials,
Philadelphia.
(14) Mazzone, H.M., and G.H. Tignor, 1976. Insect viruses:
serological relationships. Adv. in Virus Res. 20:237-270.
(15) Pagano, J.S., and E. Huang, 1974. The application of
RHX-DHX cytohybridiration to viral diagnostics. Xni Viral
Immunodiagnosis. E. Rurstak and R. Morisset, eds., Academic Press,
Inc., N.Y.
(16) Pound, J.G. 1977. Safety and potential hazards of the
entoaopathogeh Mattesia trogodermae to nontarget species. Ph.D.
Dissertation, u. Wisconsin.
(17) Reynolds, 6.J* 1978. Enzyme labelled antibody in
histopathology. Qualityline (Winter 1978/1979):2-10.
(18) Shelbourne, B.E. 1962. Experimental seawater systems
for rearing fish larvae. Pp.81-93 in Seawater Systems for Experimental
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.
(19) Smith, C.E. and M.D. Summers, 1978. Analysis of
baculovirus genomes with restriction endonucleases. Virology 89:517-
S27.
(20) Strickland, J.D.H., and T.R. Parsons. 1968. A practical
handbook of seawater analysis. Fish Res. Board Can. Bull. No.
167., 311 pp.
(21) 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.
(22) Undeen, A.E., and J.V. Maddox. 1973. The infection of
nomoaquito hosts by injection with spores of the microsporidan
Nosema alqerae. J. Invert. Path. 22:258-265.
(23) Van Essen, F.W., and D.W. Anthony. 1976. Susceptibility
of nontarget organisms to Kosema algerae (Microsporida: Nosematidae),
a parasite of Mojquitoes. J. Invert. Path. 28:77-85.
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(24) Weber, £.K.
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(b) Test standards. The plant studies mm outlined in this
••etien should meet the Subdivision J general test standards
(§ 120-3} and specific tast standards [$$ 121-Kb), 122-1 (b), and
122-2(b)] for th« appropriate tests with the following exceptions.
(1) Test substance. A typical end-use product shall be
tested.
(2) Dose levels. One concentration level equal to no less
than the "^ri""^ label rate shall be tested. The phrase "the
••--(•mi. label rate" Mans 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-inch column
of water.
(3) Additional plants. In addition to the plant species
identified in Subdivision J (5 122-1 and -2), five species of the
same genus or, if not available, of the same family should be tested
in order to evaluate the selectivity of the mierobial agent. The
species should be of economic importance such as horticultural or
agronomic crops, or vegetation useful to domestic or wild animals.
(c) Reporting. In addition to the general information outlined
in Subdivision J [) 120-4(b)], the reporting requirements for the
other tests lH 121-1{c), 122-1(c), and 122-2(c)] should be followed.
(d> Tier progression, (i) If an adverse effect or response
on plan-c growth and development is 25 percent or greater for
terrestrial plants and 50 percent or greater for aquatic plants
with respect to the control, testing at Tier II (Environmental
Expression, §f 155-15 through 155-23) is required as specified
in 40 C7R J 158.165.
(ii) If less than a 25 percent adverse effect or response for
terresrrial plants or 50 per cent for aquatic plants is noted, no
additional testing at higher tiers Is ordinarily necessary. The
Agency, after review ing the data, may recommend certain additional
tests to determine a more accurate no observed effect level.
154-23 Itentarget insect testing for toxicitv/pathogen!city to
insect predators and parasites; Tier I.
(a) When required. Data on the toxieity/pathogenicity of a
mierobial pest control agent are required by 40 CFR § 156.165 to
support the registration of each end-use product containing a
aicrobial pest control agent intended for outdoor application when
the proposed use pattern indicates that insect predators and/or
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parasite* may be exposed to th« pesticide/ and each manufacturing-
use produce that legally may be used to formulate such an end-use
product* .See 40 CFR J 158.50 and § 158.165 to determine whether
these data oust be submitted; Section II-B of this subdivision
contains an additional discussion of the f orsailators' execution
and who, as a general rule/ is responsible for submission of the
required data.
(b) Test standardst In addition to satisfying the applicable
general test standards outlined in $ 150-3 of this subdivision,
this study should meet the following standards:
(1) Test substance. Data must be derived fro* testing con-
ducted with the technical grade of each active ingredient in the
product;
(2) Test species. Testing should be performed on three species
of insects, representing three of the following groups:
Predaceous hemipterans
Predaceous coleopterans
Predaceous mites
Predaceous neuropterans
Parasitic hymenopterans
(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; and
(4) Duration of test. Control and treated insects should be
observed for at least 30 days after dosing.
(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) Non-genetieally engineered
microorganisms. (i) 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 OTR $ 158.165.
(11) If toxic or pathogenic effects are not observed in this
study, additional testing is ordinarily not necessary.
(2) Genetically-engineered microorganisms. Testing at the
Tier II level is recommended for all genetically-engineered micro-
organisms, regardless of the outcome of results in Tier I.
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154-24 Honey bee toxicity/pathoqenicitv test; Tier I.
(a) When required. Data on the toxicity/pathogenicity of a
microbial pest control agent are required by 40 CFR § 158.165
to support the registration 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 manufac-
turing-use product that legally may be used to formulate such an
end-use product. See 40 CFR § 158.50 and § 158.165 to determine
whether these data must be submitted? Section II-B of this subdivi-
sion contains an additional discussion of the formulators' exemp-
tion and who, as a general rule/ is responsible for submission of
the required data*
(b) Test standards. In addition to satisfying the applicable
general test standards outlined in § 150-3 of this subdivision, this
study shall meet the following standardst
(1) Test substance. Data must be derived from testing con-
ducted with the technical grade of each active ingredient in the
product.
(2) Test species. Testing shall be performed on the honey
bee, Apis me1lifera.
(1) 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 require-
ments are the same as those specified in $ 150-4 of this subdivision.
(d) Tier progression. (1) Mon— genetically encr^^eered **^ cro—
organisms, (i) 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 potential for adverse effects exists/ Tier
II testing will be required as specified An-40 CFl4»W«.1«5.
(ii) If toxic or pathogenic effects are not observed in this
study, additional testing is ordinarily not necessary.
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(2) Genetically-engineered microorganisms. Testing at the
Tier II level is recommended for all genetically-engineered micro-
organisms, regardless of the outcome of Tier I tasting.
(a) References. Tha following references are provided for
use in the development of acceptable test protocols for conducting
a honey bee toxicity/pathogenicity tast with a microbial pest
control agant:
(1) Davidson/ W.W., H.L. Morton, J.O. Moffatt/ and S. Singer.
1977. Effact of Bacillus aphaarieus strain SSII-1 on honey bees/
Apis mallifara. J. Invert. Pathol. 29:344346.
(2) Menapace, O.K., R.R. Sackett, and W.T. Wilaon. 1978.
Adult honey bees are not susceptible to infection by Nosema locustaa.
J. Econ. Entomol. 71:304-306.
(3) Morton, B.L., J.O. Moffatt/ and F.D. Stewart. 1975.
Effect of alfalfa looper nuclear polyhedrosis virus in honey bees.
J. Invert. Pathol. 24:139-140.
Group B-2: Tier III Testing.
§ 154-25 Terrestrial wildlife and aquatic organism toxieity
tasting; Tiar III.
(a) Whan required. Tha data outlinad in saction sarias 71
and 72 of Subdivision E ara required by 40 CFR { 158.165 to support
tha registration of aach and-usa product and aach manufacturing-use
product that nay be legally used to formulate such an end-use
product when toxic effects on nontargat terrestrial wildlife or
aquatic organisms ara reported in one or more Tier I teats
(§§ 154-16 through -21) and results of Tier II tests (section
series 155) indicate exposure of the microbial agant to tha
affected nontargat terrestrial wildlife or aquatic organisms.
See 40 CFR $ 158.50 and $ 158.165 to determine whether these data
must be submitted; Saction II-B of this subdivision contains an
additional discussion of the femulators' exemption who/ as a
general rule, is responsible for submission of tha required data.
(b) Test standards. The test standards are the same as
those found in $$ 71-1 through -5 and 72-1 through -6 of Subdivi-
sion E.
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(c) Reporting and •valuation of data* The reporting «nd
•valuation .provisions are the saae AS those found in $J 71-1
through -5 and 72-1 through -6 of Subdivision E.
(d) Tier progression* Farther testing shall b« required as
specifi«d in 40 crx § 158.165 and outlined in §f 71-1 through -5 and
72-1 through -6 of Subdivision S.
J 154-26 Long-tern avian pathooenieity and reproduction test;
Tier III,
(a) When required. Data on the long-term avian pathogenieity
and reproduction effects of a microbial pest control agent are re-
quired by 40 CFR $ 158.165 to support the registration of each end-
use product intended for outdoor application and each manufacturing-
use product that legally Bay be used to formulate such an end-use
product when:
(1) Pathogenic effects are observed in Tier I ($} 154-16 and
-17) at a level equal to the adjusted host equivalent amount t or
(2) Chronic, carcinogenic, or teratogenlc effects are reported
in tests outlined by H 152-53, -54, and -56, respectively, for
evaluating hazard to humans and domestic animals; and
(3) Environmental expression testing (f§ 155-1S through -20)
indicates that exposure of terrestrial animals to the microbial
agent is likely.
(4) See 40 CFR § 158.50 and § 158.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the f emulators' exemption
and who, as a general rule, is responsible for submission of the
required data.
(b) Test standards. Data should satisfy the general test
standards in $ 150-3 of this subdivision, and the following test
standards t
(1) Test substance. Data shall be derived from testing
conducted with the technical grade of each active ingredient in the
product.
(2) Species . Testing should be performed on one avian
species (preferably the bobwhite quail or mallard duck). The
species selected should be the same as that selected for the
avian injection pathogenieity test in $ 154-17.
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(3) Age. Birds Approaching their first breeding season should
be used.
(4) Controls. A concurrent control group is recommended and
should be treated with the technical grade of the active ingredient
containing inactivated aicrobial agent.
(5) Concentration levels* At least two treataent levels
should 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) Number of birds per treatment group. Each treatment
group should be replicated. For bobwhite quail and mallard ducks,
a minimum of 12 pen replicates should be used.
(7) Duration of exposure. Birds should be exposed to treated
diets beginning not less than 10 weeks before egg laying is expected,
and extending throughout the laying season.
(c) Reporting and evaluation of data. In addition to the
information specified in § 150-4 of this subdivision, the test
report should contain the following information:
(1) Test results. The following information should be reported
for all test groups:
(i) All observed abnormal behavior;
di) All observed morphological and physiological responses;
(111) Time and date of mortalities;
(iv) Results of gross necropsy tests conducted on all birds
dying before termination of the test and on a representative
sample of those that survived;
(v) Any evidence of multiplication of microbes (e.g.,
lesions) in selected body tissues that would normally be affected
by an infection including the liver, kidney, lungs, spleen, cere-
brospinal system and gastrointestinal tract.
(vi) Description of the method chosen to assess the cause and
effect of any lesions noted;
(vii) Morbidity;
(viii) Accidental deaths or injuries;
(ix) Observable clinical signs; and
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.(x) Clinical tests.
(2) Test conditions. The following information should be
reported for treated and untreated test groups:
(i) Species;
(ii) Strain;
(111) Age;
(iv) Body weight;
(v) Number of birds per test (include sex ratio);
(vi) individual identification of birds;
(vii) Diet;
(viii) Storage;
(ix) Feed consumption (grams per day);
(x) Observation on palatability or repellency;
(xi) Housing conditions of test birds:
(A) Space allocations for mating, nesting;
(3) Measurements taken to insure that the birds were pro-
tected from injuries;
(C) Lighting program, including hours per day and wattage
or footcandles at bird level;
(xii) Diagram of test layout:
(xiii) Temperature;
(xiv) Water supply; and
(xv) pretest and test history or medical and chemical admini-
stration.
(3) Egg and hatching data* The following information should
be reported for each treated and untreated test group:
(i) Egg shell thickness;
(ii) Cracked eggs;
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', iii) Eggs laid (number eggs per bird per day and per season);
(iv) Hatching egg storage data:
(A) Temperature;
(B) Humidity;
(C) Incubation data;
(D) Eggs seti and
(E) Egg-turning frequency;
(v) Fertility (viable embryos);
(vi) Live 3-week embryos;
(vii) Embryos that mature/ embryos that pip shell/ embryos
that liberate themselves/ and a determination of hatchability ;
(viii) Dead embryos;
(ist) Fourteen-day-old survivors;
(x) Crippled survivors;
(xi) Post-hatchling mortality;
(xii) Weights of fourteen-day-old survivors; and
(xiii) Any signs of pathogenic effects in poct-hatchling sur-
vivors.
(4) Pesticide test data. The levels of concentration of
the nicrobial pest control agent in the feed and the rationale
for choosing such levels should be reported.
(d) Tier progression. (1) If pathogenic effects are observed
at actual or expected exposure levels:
(i) The applicant should reconsider the proposed registration
of the product; and
(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 (§ 154-33} may not be feasible. If adequate constraints or
quarantine methods are possible, testing at Tier IV is required as
specified by 40 CFR § 158.165.
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12) If no pathogenic effects are observed at actual or ex-
pected field residue exposure levels, no additional testing is
ordinarily necessary.
(e) References. The following references are provided for
use in the development of acceptable test protocols for conducting
long-tern avian pathogenicity and reproduction tests with microbial
pest control agentst
(1) Heinz, G. H. 1976. Methylaercury: Second year feeding
effects on mallard reproduction and duckling behavior. J. Wildl.
Manag. 40(1):82-90.
(2) Heinz, C.H., and L.N. Locke. 1976. Brain lesions in
mallard ducklings from parents fed methylmercury. Avian Diseases
20<1):9-17.
(3) U.S. Environmental Protection Agency, Registration of
Pesticides in the United States: Proposed Guidelines, Subpart E—
Hazard Evaluations Wildlife and Aquatic Organisms. 1978 (July 10).
Fed. Reg. 43(132):2972929731.
§ 154-27 Definitive aquatic animal pathogenicity tests; Tier III.
(a) When required. Data from definitive pathogenicity tests
with fish and/or aquatic invertebrates are required by 40 CFR § 158.165
to support the registration of each end-use product intended for
use in water or expected to be transported to water from the intended
•ase site, and when pathogenicity or infectivity was observed in
Tier I tests and to support the registration of each manufacturing-
use product that legally may be used to formulate such an end-use
product. See 40 CFR § 153.50 and $ 158.165 to determine whether there
dara must be submitted; Section II-B of this sudivision contains an
additional discussion of the fomulators* exemption and who, as a
general rule, is responsible for submission of the required data.
(b) Test standards. Data should be derived from tests that
satisfy the general test standards in § 150-3 of this subdivision,
and the test standards in Tier I {$§ 154-19 and -ZOIT'^with the
following exceptions:
(1) Test substance. Data should be derived from testing
conducted with the most challenging form of each active ingredient
(microorganism) in the product, as determined from results of Tier
I testing.
(2) Test organisms. Testing should be conducted on one or
-ere of the fallowing types of species depending upon results of
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Tier I tests, intended use sites, and estimated environmental
concentrations:
(i) Freshwater fish (e.g., rainbow trout);
(ii) Freshwater aquatic invertebrate;
(iii) Estuarine or marine fish (e.g., sheepshead minnow);
and/or
(iv) Estuarine or marine invertebrate (e.g., shrimp).
(3) Method of pesticide administration. The test substance
should 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.
(c) Reporting and evaluation of data, The provisions in
Tier I, §§ 154-19 and -20, apply.
(d) Tier progression, (i) If pathogenic effects are
observed, further testing at Tier IV (§ 154-29) may be specified
in 40 CFR § 158.165.
(ii) If pathogenic effects are not observed, additional
testing at higher tiers is ordinarily not necessary.
(e) References. Refer to paragraph (e) in §§ 154-20
and -21.
§ 154-28 Fish embryo-larvae studies and life cycle studies of fish
and aquatic invertebrates; Tier III•
(a) When required. Data from fish embryo-larvae studies and/
or fish life cycle studies and/or aquatic invertebrate life cycle
studies are required by 40 CFR § 158.165 to support the registration
of each end-use product intended for use in water or expected to
be transported to water from the intended use site, and when
pathogenieity or infectivity was observed in Tier I tests and to
support the registration of each manufacturing-use product that
aay be legally used to formulate such an end-use product. See 40
CFR S 158.50 and $ 156.165 to determine whether these data must be
submitted; Section II-B of this subdivision contains an additional
discussion of the formulators' exemption and who, as a general
rule, is responsible for submission of the required data.
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satisfy the general test standards in § 150-3 of this subdivision,
ar.d the following test standards:
(1) Test substance. Data shall be conducted with the most
challenging fora of each active ingredient (microorganism) in the
product/ as determined from results of Tier I tests (§§ 154-19 and
-20) or other Tier III tests (}§ 154-27, -29, and -30).
(2) Duration of test, (i) Fish embryo-larvae test. The
eabryolarvae test requires that aquatic organism* be exposed to the
test substance during the embryo-larval phase (e.g., a fish "egg-fry"
test), but not during all stages of life-cycle of one generation of
the test species.
(ii) Aquatic invertebrate and fish life-cycle tests. The
aquatic invertebrate and fish life-cycle tests require that the
test animals be cultured in the presence of the test substance from
egg to egg or from one stage of the life cycle to the same stage of
the next generation.
(3) Test organisms and methods. The applicant should consult
with the Agency regarding the appropriate species and test methods.
The choice of species and teat methods may have to be tailored to
the microorganism's characteristics.
(c) Reporting and evaluation of data. In addition to the
information specified in § 150-4 of this subdivision, the test
report should contain the following information (when appropriate)
on the nontarget test organism:
(1) Reproductive effects;
(2) Detailed records of spawning, egg numbers, fertility, and
fecundity;
(3) Estimated no observed effect level;
(4) Mortality data;
(5) Statistical evaluation of effects;
(5) Locomotion, behavioral, physiological, and pathological
effects;
(7) Definition of the criteria used to determine effects;
(8) Summary of observed signs of pathogenicity or other effects;
(2) Verification of micro-organism(s) responsible for any
observed pathogenic effects; and
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; 13) TStage .of life cycle in which organisms were tested.
(d) Tier progression. (1) If pathogenic effects are observed,
further testing at Tier IV, at § 154-34, may be required as specified
in 40 CFR $ 158.165.
(2) If no pathogenic effects are observed, additional testing
at higher tiers is ordinarily not necessary.
(e) References. The following may contain useful background
information for developing acceptable protocols:
(1) Fish early-life stage;
(i) National Water Quality Laboratory Committee on Aquatic
Bioassays. 1971. Recommended bioassay procedure for fathead
minnow Pimephales promelas (Rafinesque) chronic tests. National
Water Quality Laboratory. Duluth, Minn. 13 pp. (Revised January
1972.)
(ii) _ 1971. Recommended bioassay procedure for
brook trout Salvelinus ffontinalis (Mitchell) partial chronic tests.
national Water Quality Laboratory, Duluth, Minn. 11 pp. (Revised
January 1972).
(iii) Hansen, D.J., P.R. Parrish, S.C. Schimmel, and L.R.
Goodman. 1978. Lifecycle toxicity test using sheepshead minnows
:Cyprinodon variegatus). Pp.109-116 in Bioassay Procedures for
the Ocean Disposal Permit Program. U.S. Environmental Protection
Agency, Office of Res. and Dev. EPA 600/9-78-010.
(2) Fish and aquatic invertebrate life-cycle tests;
(i) Biesinger, K.E. 1974(a). Procedure for Daphnia magma
tests in standing system. U.S. Environmental Protection Agency,
Environ. Res. Lab., Duluth, Minn.
(ii) Biesinger, K.E. 1974(b). Procedure for Daphnia magna
chronic tests in flowing system. U.S. Environmental Protection
Agency, Environ. Res. Lab., Duluth, Minn.
(iii) Hansen, D. J., P. R. Parrish, S. C. Schimmel, and L. R.
3oo±r.ar.. 1978. Life-cycle toxicity test using sheepshead minnows
(Cyprinodon varieqatus). Pp. 109-116 in Bioassay Procedures for the
Ocean Disposal Permit Program. U.S. Environmental Protection
Agency, Office of Res. and Dev. EPA-600/978-010.
(iv) Niamo, D.E., 7.L. Hamaker, and C.A. Sommers. 1978. En-
tire life-cycle toxicity test using my»ids (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.
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264
(v) Sehimmel, S.C., and D.J. Hansen, 1974. Sheepshead min-
now Cvorinodon variegatus: an estuarine fish suitable for chronic
(entire life-cycle) bioassays. Proc. 28th Ann* Cong. S.E. Assoc.
•3aae-Fish Coom. Pp. 392-398.
(vi) National Water Duality Laboratory Committee on Aquatic
Bioassays. 1971. Recommended bioassay procedure for fathead
minnow Pimephales promelas (Rafinesqui) chronic tests* National
Water Quality Laboratory, Duluth, Minn. t3 pp. (Revised January
1972.)
(3) Additional information Additional information may be
found in the following reference:
(i) Biesinger, K.C. 1974(c). Culturing methods for Daphnia
and certain other cladocerans* U.S. Environmental Protection
Agency, Environ. Res. Lab., Duluth. Minn*
§ 154-29 Aquatic ecosystea-pathoqenieitv tests; Tier III.
(a) When retired. Data from aquatic ecosystem-pathogenicity
tests are required by 40 CFR § 158*165 to support the registration
of each end-use product intended for outdoor application and
each manufacturing use product that legally may be used to formu-
late such an end-use product, if, after an analysis of the micro-
bial agent's properties/ the individual us* patterns, and the
results of previous nontarget organism and environmental expres-
sion 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 agent 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:
(1) Infectivity or pathogenicity demonstrated in previous
testing; and
(2) Viability of the microorganism in natural waters as demon-
strated in Tier II tests.
(3) See 40 CFR $ 158.50 and J 158.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the femulators' exemption and
who, as a general rule, is responsible for submission of the required
data.
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-265
(b) Test standards. Specific standards will be establish-
ed -.on * -case-by-case basis. Data sufficient to satisfy the general
test 'standards in § 150-3 of this subdivision, and the following
test standards:
(1) Test substance. Data shall be derived from testing con-
ducted with the most challenging form of each active ingredient
(microorganism) in the product/ as determined from results of Tier
I tests or any other Tier III tests.
(2) Test organisms, (i) Following consultation with the
Agency, the registration applicant should choose one or more of
the following species to be used in aquatic ecosystem testing:
(A) A typical bottom-feeding fish («.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., shrimp, or cray fish); or
(E) Nymphs (e.g., mayfly).
(c) Reporting and evaluation of data. In addition to the in-
forraation outlined in § 150-4 of this subdivision, specific data
reporting and evaluation provisions will be established on a case-
by-case basis following consultation with the Agency.
(d) Tier progression. (1) If pathogenic effects are observ-
ed then simulated and actual field testing may be required by 40
C?a § 158.165 and specified in § 154-33.
(2) If no pathogenic effects are observed, additional testing
at higher tiers is ordinarily not necessary.
(e) References. The following may 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 C14-pesticides by bluegill sunfish
during continuous exposure. Pp. 119-142 in Structure-activity cor-
relations in studies of toxicity and bioconcentration with aquatic
crsanisns. 3.D. Veith and D.E. Konasevich, eds. Proceedings of
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266
a 5vaposi.ua, 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) Schiamel, B.C., J.M. Patrick, Jr., and A.J. Wilson. 1977.
Acute toxlcity to and bioconcentration of endosulfan by estuarine
animals* Pp. 241-252 £n Aquatic Toxicology and Hazard Evaluation.
F.L. Mayer and J.L. Hamalinx, eds. STP #634, American Society for
Testing and Materials, Philadelphia, Pennsylvania.
$ 154-30 Special aquatic tests - tissue culture,"microorganism/
stress interaction tests. [Reserved]
§ 154-31 Plant studies: Tier III.
(a) When required. Data on the effects of a microbial pest
control agent on plant growth and development are required by 40
CFR § 158.165 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 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 {§§ 155-15 through -23).
See 40 CFR $ 158.50 and § 158.165 to determine whether these data
must be submitted; Section II-B of this subdivision contains an
additional discussion of the femulators' exemption and who, as a
general rule, is responsible for submission of the required data.
(b) Test standards. The test standards are the same as
those in Tiers II through III [§ 123-1 through § 124-2] of Subdi-
vision J.
(c) Reporting. The reporting provisions are be the same
as those in Tiers II through III [§ 123-1 through § 124-2] of
Subdivision J.
(d) Tier progression. The tier progression criteria are the
same as those .n Tiers II through III [§ 123-1 through § 124-2] of
Subdivision J.
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267
§ 15-1-32 Reserved!
Group B-3: Tier IV Testing*
§ 154-33 Simulated and actual field testing for mncmntls and birds;
Tier IV.
(») When required. (1) Data on the avian and mammalian path-
ogenicity of a microbial pest control agent in the field ere required
by 40 CFR § 158.165 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 when:
(i) Pathogenic effects at actual or expected field residue ex-
posure levels are reported in Tier III; and
(ii) The Agency determines that quarantine methods will pre-
vent the microbial pest control agent from contaminating areas ad-
jacent to the test area.
(2) The Agency will determine on a case-by-case basis which
test (simulated small-pen field, simulated large-pen field test, or
fall-scale field test) shall be required.
(3) See 40 CFR § 158.50 and § 158.165 to determine whether
these data must be submitted; Section II-B of this subdivision
contains an additional discussion of the femulators' exemption
and who, as a general rule', is responsible for submission of the
required data.
(b) Test standards. Data should be derived from tests that
satisfy the general test standards in § 150-3 of this subdivision
and the following test standards:
(1) Test substances* Data shall be derived from testing con-
ducted with a typical end-use product.
(c) Reporting and evaluation of data. In addition to the
information specified in { 150-4 of this subdivision, the test
report should contain any additional information recommended follow-
ing consultation with the Agency.
(d) References. The following references are provided for
use in the development of acceptable test protocols for conducting
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263
simulated And actual field tests for mammals and birds with micro-
biai pest control agents:
(1) Simulated large and small pen field tests.
(i) Black/ C. T., and G. L. Zorb. 1965. Effect of malathion
sprays on penned pheasants. Mich. Conserv. Dept. Research and
Develop. Rpt. No. 34.
(ii) Heezen, K. L. 1973. Pesticide effects on pheasants.
job Complet. Rept. Proj. No. W-118-R-6, Job No. 119.1 Mich.
Dept. Conserv. 21 pp.
(iii) Kreitter, J. ?., and J. W. Spann. 1968. Mortality
among bobwhites confined to a heptachlor contaminated environment.
J. Wildl. Manage. 32(4): 874-878.
(iv) Zorb, G. L. 1968. Effects of pesticides on wildlife.
Job Complet. Rpt. Proj. No. W-118-R-1, Job. No.4 Mich. Dept.
Conserv. 6.
(2) Full-scale field tests for hazard to wildlife.
(i) Buckner, C. H., P.D. Kingsbury, B. B. McLeod, K.L. Mortenson,
and D. G. H. Ray. 1974. Impact of aerial treatment on non-target
organisms. Algonquin Park, Ontario and Spruce Hoods/ Manitoba. Inf.
Rep. CC-X-59, Sect. F. Chen. Control Res. Inst. Can. For. Serv.,
Ottawa/ Ont.
(ii) Buckner, C. H., B. B. McLeod, and P. D. Kingsbury. 1975.
The effect of an experimental application of nuclear polyhedrosis
virus upon selected forest fauna. Rep. CC-X-101. Chem. Control
Res. Inst., Can. For. Serv., Ottawa* Ont.
(iii) Ecological Research Committee. 1970. Recommendations
far an international standard for a mapping method in bird census
work. Pp. 49-52 _in Sympos. on bird Census and Environ. Monit.,
Bull. 9 (British Trust for Ornithology. Beech Grove; Tring;
Hertfordshire/ England).
(iv) Ernien, J. T. 1971. Population densities of birds de-
rived from transect count. The Auk 88:343.
(v) Jolly, G. M. 1965. Explicit estimates *?om capture-
recapture data with both death and immigration stochastic model.
3ionetrika 52:225-247.
(vi) Kingsbury P. B. McLeod, and K. Mortensen. 1978. Impact
of Applications of the Nuclear Polyhedrosis Virus of the Red-headed
Pir.e Sawfly, Meodiprion leeontei (Pitch), on Non-target Organisms
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25?
ir. '?•". ?.e?ort ??:'.-:<-11. ~analisr. Forestry Servire, Dept. of
ri£.-.er:.is *~..i the ir.viror*.aer.t.
'. vii) Lautenschlager, R. A., H. Rothenbacher, and J. D.
Podgwaite. 1978. Response of small mammals to aerial applications
of the nucleopolyhdro«is virus of the gypsy moth, Lymantria. disgar.
Sr.viron. Sntgmel. 7(S):676-634.
(viii) McSwen, X,. C., C. E. Knittle, and M. L. Richmond.
1972. Wildlife effects from grasshopper insecticides sprayed on
snortgrass range* J. Range Manage* 25(3): 188-194.
(ix) Swift, 0. M., and N. R. French (Coordin.) 1972. Verte-
brates - saall mammals. Pp. 24-28 in Basic Field Data Collection
Procedures for the Grassland Bioae. IBP. Nat. Res. Ecol. Lab.,
Ft. Collins, Colo. 86 pp. (Tech. Rpt. No. 145).
§ 154-34 Simulated or actual field testing for acruatic organisms;
Tier IV.
(a) When required. (1) Data from a short-term simulated
field test (where confined populations are observed), and/or an
actual short-term field test, (where natural populations are obser-
ved) ara required by 40 CFR § 158.165 to supporr the registration of
each product and each manufacturing-use product that legally may
be use*! to formulate such an end-use product that is likely to
cause adverse short-term or acute effects, based on consideration
of available laboratory data, use patterns/ and exposure races.
(2} Data from a long-term simulated field test (e.g., where
reproduction and growth of confined populations are observed) ar.d.
:r 2.-. actual field test (e.g., where reproduction and growth of
latural populations are observed) are required if laboratory data
indicate adverse long-tera, cumulative/ or life-cycle effects :nay
result fron intended use.
(3) See 40 CFK § 158.50 and § 153.165 to determine whether
these data must be submitted; Section II-B of this subdivision
zsr.tains an additional iiscussion of the fcrmulatsrs''exenpti?-.
a.-.i who, as a general rule, is responsible for submission of the
required data.
(z) Test standards. Data should be derived from tests that
satisfy the general test standards in § 150-3 of this subdivision,
ar.d the following test standards:
' 15 Test substance. Data shall be derived frara tsstir.7 ton-
•iucfsi rfith t'r.e erti—ise product.
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.270
(2) Concentration analysis. Th« concentration of the test
substance in the water should be determined at the start of the
study «nd from samples collected periodically.
(3) Test conditions* The test conditions for conducting field
tests should resemble the conditions likely to be encountered under
actual use conditions* specifically, the pesticide should be applied
at the rate, frequency, and method specified on the label.
(4) Endangered species. Studies shall not be conducted in
areas containing, or suspected to contain, threatened or endangered
plants or animals.
(5) Residue levels* When the test substance is applied under
simulated or actual field condition testing, residues should be
determined in appropriate vegetation, soil, water, sediments, and
other environmental components, and in selected tissues of test
organisms.
(6) Other standards. Universally acceptable standards for
conducting field tests are not possible because of the many mechan-
isms by which a microbial agent may enter, persist, and/or reproduce
in the environment, and the variety of food sources and habitats
that may be affected. Therefore, the standards for conducting these
tests and the information that should be reported will be established
following consultation with the Agency.
(c) Reporting and evaluation of data. In addition to the in-
formation outlined in § 150-4 of this subdivision, specific data
reporting and evaluation provisions will be established following
consultation with the Agency.
(d) References. The following may contain useful background
information for developing acceptable protocols:
(1) Anonymous. 1975. Field Testing Techniques. Fed. Regis.
40(123):26909-26912 (June 25).
(2) Buckner, C. H., P. D. Kingsbury, B. B. McLeod, K.L.
Mortenson, and 0. 6. H. Ray. 1974. impact of aerial treatment on
non-target organisms. Algonquin Park, Ontario and Spruce Woods,
Manitoba. Inf. Rep. CC-X-59, Sect. F* Chem. Control Res. Inst. Can.
For. Serv., Ottawa, Ont.
(3) Buckner, C. B., B. B. NcLeod, and P.O. Kingsbury. 1975.
The effect of an experimental application of nuclear polvhedrosis
virus upon selected forest fauna. Rep. CC-X-101. Chem. Control Res.
Inst., Can. For. Serv., Ottawa, Ont.
(4) Kingsbury, P., B. McLeod, and X. Hortensen. 1978. Impact
of applications of the nuclear polyhedrosis virus of the red-headed
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271
pin* saw-fly, Neodiorion lecontei (Fitch), on non-target organisms
in 1977. Report FPM-X-11. Canadian Forestry Service', Dept. of
Fisheries and the Environ•
(5) Mulligan, F. S., C. H. Schaefer, and 1. Miura. 1978.
Laboratory and field evaluation of Bacillus sphaerieus as a Mosquito
control agent. J. Econ. Ent« 71(5):774-777.
§ 154-35 Simulated or actual field testing for insect predators and
parasites: Tier IV. (Reserved)
154-36 Simulated or actual field testing for insect pollinators:
Tier IV. [Reserved]
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272
S«rl*S 155: rHVIRONMJENTAL PATE AND EXPRESSION GUIDELINES FOR
BIORATIONAL PESTICIDES
Subsarie* 155AJ Tisr II Enyironmaatal Guidelines for Biochemical
Pest Control Agents.
155-1 General informtion.
(a) Scop*. If results of Tier I testing are_poaitive and/or
the biochemical has an Aquatic use pattarn, than furthar tasting is
raquirad by 40 CFR $ 158.165 to evaluata potential exposure. This
section series cat* forth environmental fata guidline for biochem-
ical pesticides including guidelines pertaining to the persistence
of biochemicals and to the transport of biochemicals from the site
of application to another site or medium. If the data indicate
that significant persistence and transport of these agents in any
part of the environment occurs, such that significant exposure to
nontargat organisms could be expected, additional testing in Tier
III is necessary.
(b) Use of data. Environmental fata data will generally be
used to determine the Estimated Environmental Concentration (EEC) by
performing a simple mass-balance analysis of the pesticide• taking
into consideration the pesticide application parameters (i.e., rate,
frequency, and site of application) following initial tests that
measure transport properties. (Volatility - f 155-4, Dispenser-water
leaching $ 155-5, Vapor pressure $ 151-17, and Water solubility -
J 151-17). Where persistence testing is required (Hydrolysis -
$ 1559, Aerobic soil metabolism - $ 155-10, and Aerobic aquatic
metabolism - § 155-11, Soil photolysis - § 155-12, Aquatic photo-
lysis - $ 155-13, Adsorption/desorption - $ 155-6, and Octanol/water
partition coefficient - § 155-7), each of the transformation
processes should be expressed as a half-life for the particular
environment or a rate constant for the environmental process depend-
ing on the test. Expected environmental concentrations can then
be calculated for different times using these data and the field
application rate of the pesticide* Aquatic use pattern and non-
dispenser pesticides will require mass balance analysis following
persistence tests.
(c) Assaying for degradation of biochemicals. Environmental
fate requirements include identification of degradation products
comprising more than ten percent of the initial pesticide concentration
in the following studies: hydrolysis, photodegradation (soil, water,
and vapor phase), and aerobic soil metabolism. When these studies
are required in tests of biochemical pesticides, the very low initial
pesticide concentration and the complexity of the expected products
aay make identification difficult.
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273
(1) The following method is suggested for obtaining a suit-
able sample of the degradation products* The environmental fate
studies outlined in if 155-9, -10, -12, or -13 should be performed
at one additional high concentration• That concentration of pesti-
cide should correspond to the highest concentrations) required in
the human health effects Tier X tests. Increasing the concentra-
tions greatly in environmental fate studies may change the reaction
mechanism in some eases (e.g., second order effects). To avoid
this possibility, the rate constants (or half-lives, if the rate
constants were not determined) of the reactions at high concentration
should agree within expected experimental error with the rate con-
stants/half -lives of the reactions at standard concentration.
The determination should be run to approximately 90 percent reaction
•completion or 30 days, whichever occurs first. If there is a
difference in the two rates that exceeds the error expected for
the particular reaction, a different reaction resulting in different
breakdown products may be occurring. This method may not be used
in those situations. If the rates agree, the degradation mixture
obtained may be tested in Tier I of the hotan health effects sch«
(d) Monitoring for disappearance of bioehemicals. The Agency
will allow biononitoring for disappearance of hormones and semioche-
micals in lieu of standard instrumental analysis. Biomonitoring
is useful for quantities at the detection limits of standard methods.
Procedures will vary significantly with sites and pests, however,
in all cases, a standard curve should be run to calibrate the
method. The references listed below contain information for develop-
ing a test method.
(e) Approach. Environmental fate testing should be conducted
according to the following scheme (see Figure 11):
(1) If the biochemical has an aquatic or a combined terrestrial
and aquatic use pattern, the following tests should be performed. If
there is significant adverse exposure, Tier III testing (f 154-13)
should be performed.
(i) Octanol/water partition coefficient ($ 155-7).
(ii) Hydrolysis ($ 155-9).
(iii) Aerobic aquatic metabolism ({ 155-11).
(iv) Aquatic photolysis (§ 155-13).
(2) -If the biochemical has a terrestrial use pattern only
and is not applied in a controlled release device, the provisions
of paragraph (e)(2)(ii) of this section apply*
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274
(i) Zf the biochemical has a terrestrial use pattern only and
is applied in -a controlled release device, the Volatility (} 155-4)
and Dispenser/water leaching (f 155-5) tests should be performed. Zf
any of -the following conditions are met, then the provisions of
paragraph (e)(2)(ii) of this section applyi
(A) EEC is greater than Z£SO for terrestrial animals.
(B) BBC is greater than EC25 for terrestrial plants.
(C) KEC is greater than 1/5 LD50 for terrestrial animals.
(D) CSC is greater than 1/5 EC50 for insects.
(E) The biochemical leaches significantly from dispenser and
Tier Z tests were positive for aquatic plants and/or animals.
(ii) Zf Tier Z test results are positive for insects only and
exposure is solely through the vapor phase, the Ultraviolet-visible
absorption ($ 155-8} test should be performed. Zf there is signifi-
cant adverse exposure. Tier ZZZ (§ 154-15) testing should be per-
formed*
(ill) Zf Tier Z test results are not positive for insects only
and exposure is not primarily through the vapor phase, then the
Volatility (§ 155-4) [if not performed before], Adsorption/desorption
(§ 155-€), Octanol/water partition coefficient ($ 1S5-7), and Hy-
drolysis (( 155-9} tests should be performed.
(iv) Zf adverse effects observed in Tier Z are terrestrial
effects only, the Aerobic soil metabolism (J 155-10) and Soil pho-
tolysis (S 155-12) tests should be performed. Zf there is signifi-
cant adverse exposure, Tier ZZZ testing (§ 154-12 for terrestrial
animals or $ 15414 for terrestrial plants) should be performed.
Zf significant adverse exposure is not found, then further testing
is not necessary.
(v) Zf Tier Z tests indicate aquatic effects and the EEC is
greater than 1/10 1CSO for aquatic animals or the EEC is greater
than EC50 for aquatic plants then the Aerobic soil metabolism
(§ 155-10) and Aerobic aquatic metabolism ($ 155-11) tests should
be performed. Zf not, no further testing is necessary.
(vi) Zf exposure to sunlight is expected and molar absorbtivity
is high, then the Soil photolysis (§ 155-12) and Aquatic photolysis
(5 155-13) tests should be performed.
(vii) Zf there is significant adverse exposure, Tier ZZZ testing
(§5 154-13 and -14) is necessary.
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275
figure 11—SOMMMOf OF ENVIRONMENTAL PATE TESTING SOOXE FOR BIOCHZMICALS
!• the use pattern aquatic, or terrestrial and aquatic ?
vna I no
Octanol/Water Partition Coefficient ($ 155-7)
Hydrolysis (\ 155-9)
Aerobic Aquatic Metabolism (§ 155-11)
Aquatic Photolysis (§ 155-13}
I
Perform
mass
balance
analysis
I
Is the biochemical applied in
a controlled release device?
i •—«—«-,
I Volatility (| 155-4)
Dispenser/Hater Leaching (i 155-5)
I
Perform mass balance analysis.
Is the EEC > LCSO for terrestrial animals?
or > XC25 for terrestrial plants?
or > 1/5 LD50 for terrestrial animals?
or > 1/5 EC50 for insects?
or does biochemical leach significantly
from dispenser if Tier I tests were
positive for aquatic plants and/or animals?
YZS I »»"
If Tier I testing is positive for
insects only, is exposure primarily
through vapor phase?
—NO or K/A__JL_
No further
testing
necessary
I
Volatility (§ 155-4) [if not done beforej
Adsorption/Desorption {§ 155-6)
Octanol/Water Partition Coefficient (§ 155-7)
Hydrolysis (§ 155-9)
OV-Visible Absorption (§ 155-8)
I
Signficant adverse exposure?
Do Tier I test results indicate
terrestrial effects only?
Tier III testing
necessary (\ 154-15)
No further
testing necesatry
I
Aerobic Soil Metabolism (§ 155-10)
Soil Photolysis (§ 155-12)
1
Significant adverse exposure?
Perform mass balance analysis.
Is EEC < 1/10 LCSO for aquatic animals?
or £ XC50 for aquatic plants?
v»fi I NO
I
Tier III testing required
{§ 154-12 for terrestrial animals)
and <§ 154-14 for terrestrial plants)
I Aerobic Soil Metabolism ($ 155-10)
I Aerobic Aquatic Metabolism {§ 155-11)
I I
Mo further Is there sunlight and
testing high molar absorbtivity?
necessary |
SoU Photolysis (\ 155-12)
Aquatic Photolysis (§ 155-13)
I
Is there significant adverse exposure?
2TES I N0_
Tier III testing (§§154-13 and 14)
No further t-.«gting necessary
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276
(•f) Ref erenees.
(1) American Institute of Biological Sciences (AIBS). 1977 and
1978. Analysis of Specialized Pesticide Problems of Invertebrate
Control Agents - Efficacy T««t Methods. Volumes 1, 2, 3, 4, 8,and
10. Z7A contracts 540/10-77-001, 540/10-77-007, 540/10-77-002, 540/10-
77006, and 540/10-78-002, respectively.
(2) Am, B», X. Stadler, and S. Raucher. 1975. The
electroantennographic detector - A selective and sensitive tool in
the gas chromatographic analysis of insect pheroaones. Zeitschrift
Naturforschung 30:772-725.
(3) Mitchell, J.W., and C. A. Livingston. 1968. Methods of
Studying Plant Hormones and Growth-Regulating Substances. Agriculture
Handbook No.336. Agricultural Research Service. OSDA. Washington,
o.c.
H 155-2 and -3 fReserved]
$ 155-4 Volatility.
(a) When required. Data on the volatility of a bioeheoieal
pesticide in the environment are required by 40 era § 158.165 to
support the registration of every end-use product intended for
outdoor application and each manufacturing-use product that legally
may be used to formulate such an end-use product whenever results
of any one or more of the Tier X tests (§} 154-6 through 11) indicate
potential adverse effects on nontarget organisms and the biochemical
agent is is to be applied on land. See 40 CFR § 158.50 and § 158.165
to determine whether these data must be submitted; Section ZZ-B of
this subdivision contains an additional discussion of the formula-
tors ' exemption and who as a general rule, is responsible for
submission of the required data.
(b) Test standards. In addition to the general test standards
specified in § 160-4 of Subdivision H, the following specific test
standards applyt
(1) Test substance. This study shall be performed using a
typical end-use product.
(2) T4st procedure. Samples should be tested by combined
laboratory and field studies with volatility measurements taken at
sufficient intervals to determine 90 percent loss of the agent from
the dispenser or substrate. Field studies should be conducted under
typical meteorological conditions for the environment of expected use
pattern.
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277
t.c) Reporting and •valuation of data. In addition to the
general reporting and evaluation provisions outlined in $ 160-5
of Subdivision N, the foliating apply:
(1) Meteorologic conditions (temperature, relative humidity,
wind velocity and direction/ and cloud cover) daring the times of
exposure should be summarised. Data should be reported as micrograms
product volatilized per unit time p«r milligram product originally
applied.
(d) Tier progression* Further testing may be required as described
in § 155-1(e) and required by 40 CFR § 158.165.
(e) Details of method and reference. (1) The following is an
example of an acceptable protocol for conducting a volatility
measurement of a semiochemical.
Samples of the formulated product (microcapsules, tapes,
etc.) should be placed outdoors in an area similar to the
intended site(s) of application. Samples should be collected
at five intervals beginning at the tia* the material is placed
and continuing until 90 percent of the product is lost from
the container or 90 days, whichever comes first* To determine
this point, extract the product remaining in the dispenser
with an appropriate organic solvent and quantitate the product
in the extract in comparison with the amount of product that
can be extracted from a duplicate dispenser which has not been
exposed to the environment. In addition, samples of the dis-
penser should be laboratory tested for emission rate of the
product. For example, a closed system may be arranged ia a
controlled temperature oven equipped with an air flow-controller
allowing a flow rate of 100 ml/min, and a vapor collection
device. Vapor collection time should be 2 hours. The oven
temperature selected .should be representative of the temperature
expected at the intended site(s) of application.
(2) The following reference may provide useful background
information for developing acceptable protocols:
Bierl-Leonhardt, B.A., E.D. DeVilbiss, and J.R. Pilsner. 1979.
Rate of release of disparlure from laminated plastic dispensers.
J. geon. Int. 72(3):319-321.
§ 155-5 Dispenser-water leaching.
(a) When required. Data on the leaching of a biochemical
pesticide from a passive dispenser to the environment are required
by 40 CFR $ 158.165 to support the registration of every end-use
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278
product intended for outdoor application in such a dispenser
and each manufacturing-uae product which legally nay b« ua«d to
formulate such an end-use product, whenever results of any one or
•or* of th» Tier X tact*
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279
directly or After ••• .separation or concentration step, and
determine the ae»unt of semiochemicaKs) that ha* not leached.
Calculate the percent of semioehemical(s) that leached from
the dispenser*
$ 155-6 adsorption-desorption•
(a) When required. Data on the adsorption/dasorption of a bio-
chemical pesticide in the environment are required by 40 CFR J 158.165
to support the registration of each'end-use product intended for out-
door application and each manufacturing-use product which may legally
be used to formulate such an end-use product, whenever results of
Tier I tests indicate the need for Tier XI testing as outlined in
( 155-1(e). See 40 CFR f 156.50 and { 158.165 to determine whether
these data must submitted! Section ZX-B of this subdivision contains
an additional discussion of the f emulators' exemption who, as a
general rule, is responsible for submission of the required data.
(b) Teat standards. In addition to the general test standards set
forth in Subdivision N in §§ 160-4 and 163-1, the following apply:
(1) Test substance. Studies shall be performed using the
technical grade of the active ingredient.
(2) Test procedure. The test procedures are the same as those
specified in $ 163-1 of Subdivision N, except that the procedures
described in \ 155-1(c) may be used in place of standard instrumental
analysis for monitoring disappearance of the agents when appropriate.
(c) Reporting and evaluation of data. The provisions for
reporting and evaluation of data are the same as those specified in
§ 163-1 of Subdivision N except that a description of procedures for
monitoring disappearance of the agent and corresponding test results
may be patterned after the methods selected from those described in
§ 155-1(d).
(d) Tier progression. Further tests may be necessary as de-
scribed in § 155-1(e) and required by 40 CFR § 158.165.
§ 155-7 Oetanol/water partition coefficient*
(a) When required. Data on the octanol/water partition
coefficient-are required by 40 CFR § 158.165 to support the registra-
tion of each end-use product intended for outdoor application and
each manufacturing-use product which may legally be used to formu-
late such an end-use product, whenever Tier X tests indicate the
need for Tier XX testing as outlined in $ 155-1(e). See 40 CFR
$ 158.50 and $ 158.165 to determine whether these data must sub-
mitted ; Section II-B of this subdivision contains an additional
discussion of the f emulators' exemption who, as a general rule,
is responsible for submission of the required data*
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280
(b) Test standard*. Th« test standards are the SUM as
those set forth in $ 151-17 of this subdivision.
(e) Reporting and •valuation of data. The provisions for
reporting and evaluation of data are the same as those specified in
4 64-11 of Subdivision D and presented in { 151-17 of this subdivision.
(d) Tier Progression, farther tests may be necessary as described
in ) 155-1(e) and required by 40 CFR § 158.165.
i 155-8 Ultraviolet-visible absorption.
(a) When required. Data on the ultraviolet-visible absorp-
tion spectra of a biochemical pesticide are required by 40 CFR
§ 158.165 to support the registration of each end-use product in-
tended for outdoor application whenever results of Tier I tests
(} 154-11) indicate potential adverse effects on beneficial in-
sects and the intended route of exposure of the pesticide is
through vapor phase contact. See 40 CFR J 158.50 and $ 158.165 to
determine whether these data must submitted; Section ZI-B of this
subdivision contains an additional discussion of the femulators'
exemption who, as a general rule, is responsible for submission of
the required data*
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(d) Tier progression. Farther test* may be necessary as described
in § 153-1(•) and required by 40 CFR f 156.165,
155-9 Hydrolysis.
(a) When required. Data on the hydrolysis of a biochemical
pesticide in the environment are required by 40 CFR $158.165 to
support the registration of every end-use product intended for
outdoor application and each manufacturing-use product which may
legally be used .to formulate such an end-use product, whenever
results of Tier Z tests indicate the need for Tier ZZ testing as
described in $ 155-1(e). See 40 CFR § 158.50 and $ 158.165 to
determine whether these data sust submitted; .Section II-B of this
subdivision contains an additional discussion of the femulators'
exemption who, as a general rule/ is responsible for submission of
the required data.
(b) Test standards. The general test standards are the same
as those set forth in Subdivision K at $ 160-4. In addition, the
specific test standards in § 161-1 of Subdivision M and the following
standards apply:
(1) Test substance. Studies shall be performed using the
technical grade of the active ingredient.
(2) Test procedure. The test procedures are the same as those
specified in $ 161-1 of Subdivision N, except that the procedures
described in § 155-1(e) may be used in place of standard instrumental
analysis for monitoring disappearance of the agent when such monitoring
is necessary.
(c) Reporting and evaluation of data. The provisions for
reporting and evaluation of data are the same as those specified in
§ 161-1 of Subdivision V, except that the procedure described in
§ 155-1{c) may be used to determine the toxic effects of transform-
ation products in lieu of the identification required in this
test.
(d) Tier pregression. Further tests may be necessary as
described in f 155-1(e) and required by 40 CFR $ 158.165.
§ 155-10 Aerobic soil metabolism.
(a) When required. Data on the aerobic soil metabolism of a
biochemical pesticide in the environment are required by 40 CTR
§ 152.165 to support the registration of each end-use product intended
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for outdoor application and each manufacturing-use product which
legally may b« used to formulate such an end-use product, whenever
results of Tier Z testa indicate the need for Tier II testing as
outlined In § 155-1(e). See 40 CTR § 158.50 and $ 158.165 to
determine whether these data sust submitted; Section II-B of this
subdivision contains an additional discussion of the formulators'
exemption who, as a general rule/ is responsible for submission of
the required data*
(b) Test standards. The general test standards are the same
as those set forth in Subdivision N in H 160-4. In addition, the
test standards of § 162-1 of Subdivision N and the following standards
apply!
(1) Test substance. Studies shall be performed using the
technical grade of the active ingredient.
(2) Test procedure. The test procedures are the same as those
specified in J 162-1 of Subdivision N, except that procedures described
in | 155-1(d) may be used in place of standard instrumental analysis
for monitoring disappearance of the agent when necessary.
(c) Reporting and evaluation of data. The provisions for
reporting and evaluation of data are the same as those specified in
J 162-1 of Subdivision N, except that the approach presented in § 155-
Uc) may be used in pl»ce of instrumental identification of degradable
productst
(d) Tier progression. Further tests may be necessary as described
in § 155-1(e) and required by 40 CFR J 158.165.
$ 155-11 Aerobic aquatic metabolism.
(a) When repaired. Data on the aerobic aquatic metabolism of
a biochemical agent are required by 40 CTR § 158*165 to support the
registration of end-use product intended for outdoor application
and each manufacturing-use product that legally may be used to
formulate such as end-use product whenever Tier X test results
indicate the need for Tier XX testing as outlined in $ 155-1(e).
See 40 CTR § 158.50 and § 158.165 to determine whether these data
must submitted; Section II-B of this subdivision contains an add-
itional discussion of the femulators' exemption who, as a general
rule, is responsible for submission of the required data.
(b) Test standards. The general test standards are the same
as those set forth in Subdivision N in § 160-4. In addition, the
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test standards of $ 162-4 of Subpart N and the following
apply s
(1) Test substance. Studies shall be performed using the
technical grade of the active Ingredient.
(2) Test procedure. The procedures are the same as those
specified in { 162-4 of Subdivision K except that procedures des-
cribed in § 155-1(d) may be used in place of standard instrumental
analysis for monitoring disappearance of the agent when necessary.
(c) Reporting and evaluation of data. The provisions for
reporting and evaluation of data are the same as those specified in
$ 162-4 of Subdivision N, except that the approach presented in § 155-
1(c) may be used in place of instrumental identification of the
degradation products.
(d) Tier progression. Further tests may be necessary as described
in § 155-1(e) and required by 40 CFR § 158.165.
§ 155-12 Soil photolysis.
(a) When required. Data on soil photolysis are required by
40 CFR § 158.165 to support the registraton of each end-use product
intended for outdoor application and each manufacturing-use product
which legally may be used to formulate such as end-use product/
when Tier I test results indicate the need for Tier XI testing as
described in § 155-1(e). See 40 CFR § 158.50 and $ 153.165 to
determine whether these data must submitted} Section II-B of this
subdivision contains an additional discussion of the f emulators'
exemption who/ as * general rule/ is responsible for submission of
the required data.
(b) Test standards. The general test standards for environ-
mental fate testing of biochemical pesticides are the same as
those set forth in Subdivision N in § 160-4. In addition, the
specific test standards of $ 161-3 of Subdivision M and the follow-
ing apply:
(1) Test substance. Studies shall be performed using the
technical grade of the active ingredient.
(2) Test procedure. The procedures are those specified in
$ 161-3 of Subdivision K, except that procedures described in § 155-
1(d) may be used in place of standard instrumental analysis for
monitoring disappearance of the agent when necessary.
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284
(e) Escorting and evaluation of data. The provisions for
reporting and evaluation of data are the same as those specified
in | 161-3 of Subdivision N, except that the approach presented in
4 155-1(c) Bay be used in place of instrumental identification of
degradation products•
(d) Tier progression* Further tests may be necessary as
described in § 155-1(e) and required by 40 CFR J 158.165.
$ 155-13 Aquatic photolysis.
(a) When required. Data on the aquatic photolysis of a
biochemical agent are required by 40 CFR i 158.165 to support the
registration of each end-use product intended for outdoor applica-
tion and each manufacturing-use product which may be legally used
to foraolate such an end-use product whenever Tier I test results
indicate the need for Tier XI testing as described in § 155-1(e).
See 40 CTR $ 158.50 and § 158.165 to determine whether these data
oust submitted; Section II-B of this subdivision contains an addi-
tional discussion of the femulator*' exemption who, as a general
ruler is responsible for submission of- the required data.
(b) Test standards. The general test standards are the same
as those set forth in Subdivision N in SI 160-4. In addition, the
specific test standards of J 161-2 of Subdivision N and the following
specific standards apply:
(1) Test substance. Studies shall be performed using the
technical grade of -the active ingredient.
(2) Test procedure.• The test procedure is that which is
outlined in $ 161-2 of Subdivision K, except that procedures de-
scribed in J 155-Kd) may be used in place of standard instrumental
analysis for monitoring disappearance of the agent when necessary.
(c) Reporting and evaluation of data. The provisions for
reporting and evaluation of data are the same as those specified in
§ 161-2 of Subdivision N, except that the approach presented in § 155-
1(c) may be used in place of instrumental identification of degradation
products.
(d) Tier progression. Further tests may be necessary as
described in $ 155-1(e) and required by 40 CFR § 158.165.
§ 155-14 [Reserved]
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Subaeries 1558: TITR II CNVXROIMCNTAI. DCPRESSXON DATA REQUIREMENTS
FOR KICROBIA1 AGENTS
} 155-15 general information.
'fa) Scope. Tier II environmental expression data arc required
by 40 CTR § 158.165 when toxic or pathogenic effects are observed in
hazard testing conducted on nontarget organisms in Tier I
154-16 through -24). The Tier XI guideline* consist of tests
to determine the environmental expression of a a microbial agent
in a terrestrial environment ({ 155-18), in a freshwater environment
(} 155-19), and in a marine or estuarine environment (f 155-20).
These tests are intended to demonstrate whether a mierobial agest
is able to survive/ persist or replicate in each environment, and
thereby indicate which nontarget organisms will be exposed to the
mierobial agent, if any. A determination of the environmental
expression of a mierobial agent includes an evaluation of the
growth of the agent when introduced into a new niche as well as
an evaluation of the agents' growth pattern when its population in
its normal niche is increased (as could occur immediately after
application of a mierobial agent). This includes normal saprophytic
growth. It also includes the way a microorganism may alter its
growth habits, take advantage of new environmental conditions, or
take advantage of changes in the equilibrium of the mierobial
species which exist in a coonensal association (one species benefits
and the other is unaffacted)• Thus, the "expression" of a micro-
organism's presence may be through insertion into a new niche and
continued propagation in the new niche.
(b) Approach* (1) Environmental expression testing consists
of simulated terrestrial and aquatic applications of the mierobial
agent. Terrestrial applications are conducted in a greenhouse
environment to assess expression in soil and vegetation. Aquatic
applications are conducted in aquaria to assess expression in water
and sediment.
(2) The need for terrestrial, freshwater, or marine environmental
expression testing depends on:
- The Tier I test(s) in which adverse effects were observed; and
- The intended use pattera(s) for the mierobial agent.
Thus, testing is only needed to assess environmental expression
when susceptible nontarget species (as determined in Tier I) H 154-
16 through -24) may be exposed*
(3) The relationship between Tier I test results, proposed use
pattern, and Tier XI guidelines is summarized in Table 7.
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TABLE 7— SUMMARY OP ENVIRONMENTAL EXPRESSION TESTING AS DETERMINED
BY TIER I TEST RESULTS AMD OSE PATTERNS
Tier I Test With Positive
Results (Test Species)
Estuarine/Marine
H 154-16 and 17
Avian testing - (mallard)
(quail)
Proposed Use Pattern* for
Mierobial Agent
Terrestrial
freshwater
N/A
N/A
S 154-16
Mammalian testing
N/A
H 154-19-21
Pish testing (freshwater sp.)
(estuarine/marine sp.)
(estuarine/marine *p.)
P
EM
EM
P
EM
EM
N/A
EM
EM
}§ 154-20,-21
Aquatic invertebrate testing
(freshwater ap.)
(estuarine car marine sp.)
P
EM
P
EM
N/A
EM
§ 154-22
Terrestrial plant testing
N/A
N/A
§ 154-22
Aquatic plant testing
(freshwater sp.)
(estuaring/marine sp.)
P
EM
P
EM
N/A
EM
5§ 154-23,-24
Terrestrial inaect tasting
N/A
N/A
Net applicable. Based on results of Tier I tests and the
proposed use pattern, exposure is not expected. However, the
Agency may require such tests on an inoTvldual basis.
* T: Tests to determine expression in a terrestrial environment are necessary.
3 F: Tests to determine expression in a freshwater environment are necessary.
4 EM: Tests to determine expression in a eatuarine or marine environment
are necessary.
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(4) Genetically-engineered microbial pest control agents are
subjected to a more rigorous testing scheme. Such agents should be
evaluated by the applicable -tests outlined in Tier ZI regardless of
the results observed in Tier Z testing. If adverse effects are
observed in Tier I, testing should proceed as described in the preced-
ing paragraph (as summarized in Table 7). However, if no adverse
effects are observed/ testing should be conducted in the simulated
environment(s) (terrestrial/ freshwater/ marine or estaurine)
where the exposure of nontarget organisms is expected based on the
proposed use pattern.
(e) References. The following general references contain
information useful in developing environmental expression tests for
microbial agents in terrestrial, freshwater, estuarine, and marine
environments as outlined in H 155-18 through -20:
(1) Bullock, H.R., J.P. Bollingsworth, and-A.M. Hartstack.
1970. Virulence of Heliothis nuclear polyhedrosis virus exposed to
monochromatic ultraviolet radiation. J. Invert. Path. 16:419-422.
(2) Surges, H.D., S. Hillyer, and D.O. Chanter. 1975. Effect
of ultraviolet and gamma rays on the activity of delta-endotoxin
protein crystals of Bacillus thuringienais. J. Invert. Path. 25:5-
9.
(3) Chancey/ G./ W.C. Yearian, and S.Y. Young. 1973. Pathogen
mixtures to control insect pests. Arkansas Farm Res. 22:9.
(4) Forsberg, C.W., N. Henderson, E. Henry, and J.R. Roberts.
1976. Bacillus thurinqiensist Zts Effect on Environmental Quality.
Publication, National Research Council Canada. No. 15383. 135 pp.
(5) Hostetter, D.L. and C.M. Zgnoffo, eds. 1977. Environmental
Stability of microbial insecticides. Misc. Publ. Entomol. soc. Am.
10(3): 1-117.
(6) Zgnoffo, C.M./ W.C. Yearian, S.Y. Young/ D.L. Hostetter/
and D.L.Bull. 1976. Laboratory and field persistence of new commercial
formulations of the Heliothis-nucleopolydedrosis virus/ Baculovlrus
hellothia. J. Eeon. Entomol. 69(2)»233-236.
(7) Zlnytzky, S., J.R. NePhee, and J.C. Cunningham. 1977.
Comparison of field-propagated nuclear polyhedrosis virus from
DouglasPlr Tussock Moth with laboratory-produced virus. Pacif. For.
Res. Centr. Victoria, B.C., Canada. Bi-monthly Res. Notes 33, 1.
Pp. 5-6.
(8) Krleg, A. 1975. Photoprotection against inactivation of
Bacillus thuringienais spores by ultraviolet rays. J. Invert. Path.
25(21:267-268.
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288
(9) JUlmakoff, J., and S.W. More. 1975. The ecology of
nucleopolyhedrosis virus in porina (Wiseana spp.) (Lepidoptera:
helialidae). Mew Zeal. Entoael. 6(1):73-76.
(10) Xoltia, Y., and X. Chorin-Dirsch. 1971. Alteration of
fungal morphology induced by a substance from Bacillus cereu. J.
On. Microbiol. 66: 145-151.
(11) Lewis, Franklin B. 1975. Dosage effect on target insect
populations (short- and long-tea). Selected papers from EPA-OSDA
Working Symposiaa. M. Sumaars, R« Englar, L. Falcon, P. Vail, eds.
American Society for Microbiology. Washington, D.C.
(12) Maddox, J.v. 1973. The persistence of Micrcsporida in
the environment. Enteaol. Soe. Aaer. Misc. Publ. 9:99-104.
(13) Martignoni, M.E. and P.J. Iwai. 1977. Thermal inactivation
characteristics of two strains of nucleopolyhedrosis virus (Baculovirus
subgroup A) pathogenic for Oroyia pseudetsugata. J. Invert. Path.
30: 255-262.
(14) Manjmath, D. and S.B. Mathad. 1978. Tempera tare
tolerance, thermal inactivation and ultraviolet-light resistance
of nuclear polyhedrosis virus of the araywom, Mythiana separata
(Walk) (Lepidoptera; Noetuidae). (English). Zeitsehrift Fur
Angewandte Entoaologie 87:82-90.
(15) Michael, A.H., and P.E. Nelson. 1972. Antagonistic ef-
fect of soil bacteria on rosarium-Roseum c^^rum from carnation.
Phytopathology 62:1052-1056.
(16) Mitrofanov, V.B. 1976. Effect of suboptiaua temperature
on the activation of latent virus infection of granulosis in the
codling moth. Biull. Vses Hauchno Issled Inst Zasheh Rast 37:7-10.
(Eng. sua.}
(17) Yendol, W.G. and R.A. Hamlen. 1973. Ecology of entomo-
genous viruses and fungi. Regulation of Insect Populations by
Microorganisms. Ann. N.Y. Aead. Sei. 217:18-30.
(18) Young, 8.Y. and W.C. Yearian. 1974. Persistence of
Heliothis HPV on foliage of cotton, soybean, and tomato. Environ.
Entomol. 3(2): 253-255.
S 155-16 General test standards.
(a) Applicability. This section outlines the general test
standards that apply to the studies in §$ 155-18 through -20.
Applicants for registration should also con ply with the specific test
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289
standard* established for the particular test b«ing conducted. In
the case of conflict between general and specific test standards, the
latter shall govern.
(b) Test standards. Data satisfying the provisions of { 155-
17 should neet the general test standards in Subpart N ($$ 160-4 through
-6), with the following exceptions:
(1) Microbial agent identification and quantification. The
most specific available standard methods for the identification and
quantification of the microbial pest control agent should be used.
The methods used should be consistent with those in § 150-20, Product
analysis*
$ 155-17 Reporting and evaluation of data.
(a) Results. (1) Data should be collected to determine whether
the microbial agent is able to survive/ persist or replicate in the
environment under test. This data should be expressed in the form of
a population growth or decline curve for the microbial agent. Any
other applicable method of expressing the expression of the microbial
agent population may also be used.
(2) Test reports should also contain the information designated
in following list, modified as necessary to be applicable to the
microbial pest control agent being tested* This information should
be given in sufficient detail to adequately define growth charac-
teristics of the test organism.
(i) pH and temperature for optimal growth, and the ranges of
pH and temperature within which the microorganism can survive and
grow.
(ii) Essential nutrients for growth:
(A) Carbon source (COj, carbonate, other);
(B) Minerals;
(C) Organic compounds; and
(D) Cofactors.
(iii) Potential for autotrophic growth (i.e., growth expected
in the absence of a particular facultative growth requirement).
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290
(iv) Growth response to variations in salinity.
(v) Response to known antagonista:
(A) Chemical;
(B) Biological) and
(C) Oth«ra.
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291
an additional discussion of the formulators* exemption and who, as
a general rule, is responsible for submission of the required data.
(b> Test standards* (1) Method* (i) Tests shall be conducted
in a greenhouse environment to determine whether the microblal agent
is able to survive, persist and replicate in a terrestrial environ-
aent 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 microbial agent popu-
lation:
(A) Temperature;
(B) Humidity;
(C) Precipitation (amount, frequency, pH);
(D) Sunlight;
(B) pB (soil and foliar surfaces);
(F) Nutrients (soil, vegetation).
(ii) The values selected for each parameter listed in paragraph
(b)(l)(A) through (T) 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,
sunlight, and nutrients) may supplement the greenhouse study des-
cribed in paragraph (b)(l)(i). Laboratory studies may demonstrate
that the microbial agent will be unable to sur 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 § 155-17.
(d) Tier progression. If results of this study indicate that
the microbial agent is able to persist in the terrestrial environment
such that the susceptible non-target organisa(s) tested in Tier I are
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292
likely to be exposed, then the appropriata testing in Tier XII (§§
154-25, -26, or -31) is required as specifiad in 40 CFR § 158.165.
(e) References. The following references contain information
for developing acceptable protocols.
(1) Anthony. O.W., X.K. Savage, B.X. Hazard, S.W. Avery, K.O.
Boston, and S.W. Oldacre. 1978. Yield test with Kosema alqeraa
Vavra and Undaen (Microsporida, Hosematidae) against Anopheles
albimanus Wledemann in Panama. Miseel. Publ. Kntomel. 8oe. Amar.
11:17-28.
(2) Cunningham, J.C. 1970. Persistence of the unclear poly-
drosis virus of the eastern hemlock looper on balsam foliage.
Insect Pathology Res.Institute. Sault Ste. Marie, Ontario, Canada.
Bi-monthlv Res. Motes 26:24-25.
(3) Xlgee, X. 1975. Persistence of a virus of the white-marked
tussock moth on balsam fir foilage. Maritime* Forest Res. Centra.
Predericton, Mew Brunswick, Canada. Bi-monthly Res. Motes 31:33-34.
(4) Grison, P., D. Martouret, B. Servaia, and M. Devriendt.
1976. Mierobial pesticides and environment. Ann. Zool. Eeol. Anin.
8(2):133-160.
(5) Earcourt, D.J. 1968. Persistence of a granulesis virus
of Piaris rapaa in soil. J. Invart. Path. 11:142-143.
(6) HuJeuhara, T., and a. Namura. 1972. Distribution of a
nuclaarpolyhedrosis virus of the fall webwoxm, Hyphantria cunaa, in
soil. J. Invert. Path. 19:308-316.
(7) Ignoffo, C.M., C. Garcia, D.L. Hostattar, and R.E. Pinall.
1978. Stability of conidia of an entomopathogenic fungus, Homuraea
L, in and on soil. Environ. Entomol. 7(5):724-727.
(8) Ignoffo, C.M., G. Garcia, D.L. Boatetter, and R.E. Pinnell.
1977. Vertical movement of conidia of Momuraaa rilayi through sand
and loam soils. Environ. Bntomol. 7(2):270-272.
(9) Jaques, R.P. 1967b. The persistence of a aaclear-polyhe-
drosis virus in the habitat of the host insect, Trieheplusia ni.
11. Polyhedra in aoil. Can. Entemel. 99:820-829.
(10) Jaques, R.P. 1969. leaching of the nuclearpolyhedrosis
virus of Trieheplusia ni from soil. J. Invart. Path. 13:256-263.
(11) Jaques, R.P. 1974a. Occurrence and accumulation of
viruses of Trieheplusia ni in treated field plots. J. Invert. Path.
23:140-152.
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293
(12) Jaques/ R.P. 1974b. Occurrence and accumulation of the
granule*!,* virus of Pieris rapae In treated field plots* J. Invert.
Path. 23i351-359.
(13) Xerr, X. 1974. Soil microbiological studies on
teriun radiobaeter and biological control of crown gall. Soil
Sci. 118(3):168-172.
(14) Ladd, T.L., Jr., and P.J. McCabe. 1967. Persistence of
spores of Bacillus pooilliae, the causal organism of Type A milky
disease of Japanese beetle larvae in Mew Jersey soils* J. Eeon.
rntonol.60(2)t493-49S.
(15) Lingg, A.J., and X.J. MsMmhon. 1969. Survival of
lyophilired Bacillus popilliae in soil. Appl. Mierobiol. 17:718-720.
(16) Kilner, R.J., and G.G. Lutton. 1976. Metarrhitium
anisepliaet Survival of Conidia in the Soil. Proceedings of the
First International Colloquium on Invertebrate Pathology. Queen's
University at Kingston/ Canada• Pp. 428-429.
(17) Morris/ O.N. 1973. A method of visualizing and assessing
deposits of aerially sprayed insect microbes. J. Invert. Path. 22:115-
121.
(18) Narayanan/ X., X. Oovindarajan/ and S. Jayarai. 1977.
Preliminary observations on the persistence of nuclear polyhedrosis
virus of Spodoptera litura T. Madras Aqrie. J. 64(7):487-488.
(19) Pinnock/ O.K., R.J. Brand, J.C. Kilstead/ and X.L. Jackson.
1975. Effect of tree species on the coverage and field persistence
of Bacillus thuringiensis spores. Insect biological control. J.
Invert. Path. 25(2):209-214.
(20) Roone, R.E., and R.A. Daoust. 1976. Survival of the
nuclear Polyhedrosis virus of Heliothis armigera on crops and in soil
in Botswana. J. Invert. Path. 27:7-12.
(21) Thomas, X.O./ C.7. Reichelderfer/ and A.M. Beiapel. 1973.
The effect of soil pfl of cabbage looper nuclear polyhedrosis virus
in soil. J. Invert. Path. 21(1)t21-25.
(22) Vankova, J./ and M. Svestka. 1976. The field persistence
and efficacy of Bacillus thurinqiensts formulations* Biological
control of forest pests. Anz Schadlingskd Pflansensehuts. 49(3):33-
38. (Bng. sum.)
(23) Wojciechlowska/ M. / X. Kmitowa, A. Pedorko/ and C. Bajan.
1977. Duration of activity of entomopathogenic microorganisms
introduced into the soil. Pol. Ecol. Stud. 3(2)i141-148.
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(24) Young, S.Y. 1975. Pre- And post-treatment assessment of
virus levels. Pp 139*142 in Selected papers from EPA-OSDA Working
Symposium. N. Summers, R. Engler, L. Falcon, and P. Vail, eds.
American Society for Microbiology, Wash., D.c.
$ 155-19 Tests to determine expression in a freshwater environment.
(a) When required. (1) Data on the expression of a nierobial
pest control agent ia a freshwater environment are required by 40
CFR $ 158.165 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 testing (f 154-19),
(ii) Freshwater aquatic invertebrate toxicity and pathogenicity
test (§ 154-20); or
(iii) Plant studies - aquatic (§ 154-22).
(2) Data on the expression of a oicrobial pest control agent
in a freshwater environment are required by 40 CTR $ 158.165 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 testsi
(i) Avian single dose oral toxicity and pathogenicity test (§
154-16);
(ii) Avian injection pathogenicity test (§ 154-17);
(iii) Wild mammal toxicity and pathogenicity testing (§ 154-
18);
(iv) Freshwater fish toxicity and pathogenicity testing
(§ 154-19)7
(v) Freshwater aquatic invertebrate toxicity and pathogenicity
test ($ 154-20); or
(vi) Plant studies - aquatic ($ 154-22).
(3) See 40 CTR $ 158.50 and $ 158.165 to determine whether
these data must be submitted) Section II-B of this subdivision
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contains an additional discussion of the temulators' exemption
and who as -a general rule/ i* responsible for submission off the
required data.
(b) Test standards* (1) Method, (i) Tests shall be con-
ducted in a sisolated aquatic environment (e.g., aquarium with
bottom sediment) to determine whether the microbial agent is able
to survive, persist, and replicate la 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 microbial
agent populationa
(A) Temperature;
(B) pE)
(C) Nutrients*
(D) Sunlight;
(E) Oxygen content;
(F) Hardness; and
(i) of
this lection. Specialized lab studies may demonstrate that the
aicrobial agent 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 { 155-19 in
lieu of the study described in paragraph (b)(1Hi) 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 •stabllsh that the microbial
agent population Is able to maintain Itself la 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 sane as those set forth in § 155-17.
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(d) Ti«r progression. If results of this study and use pat-
terns information indicate that the microbial agent is likely to
enter and is able to persist in a freshwater environment such that
the susceptible nontarget organism(s> tested in Tier I axe likely
to be exposed, then the appropriate testing in Tier III ($§ 154-25
through -31) is required as specified in 40 CFR $ 158.165.
(e) References. The following references contain useful
information for developing acceptable protocols.
(1) Anonymous. 1975. Isipact of the use of microorganisms on
the aquatic environment* IPA publication 660-3-75*001* Technical
Publications Office, environmental Protection Agency, National
environmental Res. Center/ Corvallis, Oregon* 97330.
(2) Anthony, D.W., K.I. Savage, E.I. Hazard, S.W. Avery, M.D.
Boston, and S.W. Oldacre* 1978. Field tests with Nosema algerae
Vavra and Ondeen (Hicrosporida, Nosematidae) against Anopheles
albinamus Wiedemann in Panama. Misc. Publ. Entomol. Soe. Amer.
11:17-28.
(3) Brand, R.<7., D.E. Pinnock, K.t. Jackson, and J.E. Hilstead.
197S. Methods for assessing field persistence of Bacillus thurin-
giensis spores. J. Invert. Path. 25:199-208.
(4) Bostetter, D.L., C.M. Ignoffo, and W.B. Kaarby. 1975.
Persistence of formulations of Bacillus thurinqiensis spores and
crystals on eastern red cedar foliage in Missouri. J. Kansas
Entomol. See. 48(2)»189-193.
(5) Ignoffo, C.N., O.L. Bostetter, and R.E. Pinnell. 1974.
Stability of Bacillus thurinqiensis and Baeulovirus heliothis on
soybean foliage. Environ. Entomol. 3(1)i117-119.
(6) Kaya, H.K. 1975. Persistence of spores of Pleistophora
ichober (Onidospora: Microsporida) in the field and their application
in oicrobial control. J. Invert. Path. 26:329-332.
(7) Pinnock, D.X., R.J. Brand, and J.X. Milstead. 1971. The
field persistence of Bacillus thurinqiensis spores. J. Invert. Path.
18:405-411.
(8) Young, S.Y. 1975. Pre- and post-treatment assessment of
virus levels. Selected psp«rs from EPA-OSDA Hbrklnj, Jymposium. M.
Summers, R. Engler, L. Falcon, and P. Vail, eds. American Society
for Microbiology.
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297
§ 155-20 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 C7R J 158.165 to support the registration of each end-use pro-
duet 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 testsi
(i) Kstuarina and marine animal toxicity and pathogenicity test
<$ 154-21)1 or
(ii) Plant studies - estuarine or marine (§ 154-22).
(2) Data on the expression of a microbial pest control agent
in a marine or estuarine environment are required by 40 CFR 158.165
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 single dose oral toxicity and pathogenicity test ($
154-16);
(ii) Avian injection pathogenicity test (§ 159-17);
(iii) Estuarine and marine animal toxicity and pathogenicity
test ($ 154-21); or
(iv) Plant studies - estuarine or marine ($ 154-22).
(3) See 40 CFR i 158.50 and $ 158.165 to determine-whether these
data Dust be submitted} Section ZZ-B of this subdivision contains
an additional discussion of the formulators' exemption and who as a
general rule, is responsible for submission of the required data.
(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 microbial agent is able to
survive, persist* and/or replicate in a marine or estuarine environment
consisting of seawater or brackish water and bottom sediment repre-
sentative of the proposed use site. The following parameters should
be varied to determine their effect on the survival and growth of
the microbial agent population:
(A) Temperature;
(S) pH;
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298
(C) Nutrients;
(D) Salinityi
(C) Sunlight;
(D Oxygen content; and
(G) Turbulence.
(ii) The value* selected for each parameter lilted 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
microbial agent's growth requirements (e.g., temperature, pH, sunlight,
oxygen) may supplement the study described in paragraph (b)(1)(i) of
this section. A specialized lab study(ies) may demonstrate that the
microbial agent vill be unable to survive and persist in a marine or
estuarine environment. In such instances, the Agency will consider
this study(ies) on an individual basis to fulfill the intent of the
testing in $ 155-20 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 § 155-17.
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 revolts of this study and use pattern
information indicate that the aierobial agent is likely to enter and
is able to persist in a marine or estuarine environmes*-*uch that the
susceptible nontarget orgaaism(s) tested in Tier I are likely to be
exposed, then the appropriate testing in Tier III (§| 154-25 through
-31) is required as specified by 40 CTR § 158.165.
(e) References. Refer to § 154-19(e).
H 155-21, -22, and -23 [Reserved]
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Series 156: PRODUCT PERFORMANCE GUIDELINES fOR BIORATIOKAL PESTICIDES
§ 156-1 General provisions.
(a) Waiver of data requirements; background and policy* The
recent amendment to sec. 3(c.)(5) of FIFRA provide* that the Admini-
strator, may waive data requirement* pertaining to efficacy. This
aaendaeat 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
chemicals which have been identified as posing a risk of unreasonable
adverse effects.
(1) Efficacy data will generally only be required by 40 CFR
$ 158.165 for products of the following types:
(i) Uses of agents intended to control microorganisms infectious
to man in any area (inanimate surface) where these microorganisms
may present a health hazard; and
(ii) Uses of agents intended for control of fungal organisms
that produce aflatoxlns.
(2) Data on phytotoxicity to the target site, i.e., crops or
other desirable plants, are considered part of an efficacy evaluation
and are thus waived. [On the other hand, data on phytotoxicity to
crops or other plants that are non-target cites are «oasldered to
be data for hazard evaluation and must be submitted on a case-by-
case basis as prescribed in $§ 154-10 and -14. Data on the effects
of microbial pest control agents on nontarget plants must be sub-
mitted for all such products as described in f§ 154-22 and -31
with cross reference to Subdivision J.]
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§ 156*2 Specific provisions.
(a) The following provisions apply to all biorational pesti-
cides regardless of whether product performaaca data ar« or ar«
not waived in accordance with I 156-1{«)»
(1) Th« available information on host •pactrua ahall ba r«-
portad;
(2) Tha tUM raquirad to aehlwra tha daalrad laval of pact
control or othar product parforaanca •tandard shall ba raportad;
and
(3) Tha «lnim» affaetiva doaaga (MBD) nacaaiary to achiava tha
daairad lav«l of pact control or othar product parformanea ctandard
shall ba raportad. Tha registrant is rafarrad to Subdivision G,
Product Parforaanca* for spacific guidanca and information on data
and reporting raquiraaants*
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Series 157: EXPERIMENTAL USE PERMIT GUIDELINES FOR BIORATIOHAL
PESTICIDES
§ 157-1 Scop* and intent.
This section scries deals with the data necessary to support
the application for an experimental us* permit for a biorational
pesticide. These guidelines ar* based on FITRA sees. 5 and 40 CFR
Part 172, and they closely Batch the guidelines in Subdivision I
in many respects. For further information on scope and intent,
refer to | 110-1 of Subdivision X.
§ 157-2 [Reserved]
§ 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 biorational pesticides
as well as to conventional pesticides.
5 157-4 Specific data requirements.
(a) General. (1) The following types of data are required
by 40 CFR § 158.165 to support an application for an experimental
use permit for a biorational pesticide:
(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) Montarget organisms; refer to paragraph la) of this section;
(v) Environmental fate; refer to paragraph (f) of this section; and
(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 1, § 112-1.
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(b> Product analysis data. To support an application for an
experimental use permit, the data outlined in || 151-10 through
-18 apply to biochemical pest control agents and §§ 151-20 through
-26 apply 'to microbial pest control agents.
(c) Residue data. For biochemical pest control agents, re-
sidue data are required by 40 CFR §• 158.165 to support an application
for an experimental use permit when the product will be used on
food or feed crops or when its use is expected to result in residues
in or on food or feed and for either of the following situations:
(1) The rate of biochemical pest control agent application
exceeds 50 grams active ingredient per acre per application; or
{2} Tier I toxicology studies conducted under paragraph (d)
of this section or under section series 153 of this subdivision
indicates a potential for human, hazard. Residue data requirements
will be determined on an individual basis for biochemical! applied
directly to food or feed, and for biochemieals whose application
rate can not be expressed in ounces per acre per application. In
these situations, the data necessary to obtain a temporary tolerance
(see Subdivision 0, Chemistry Requirements: Residue Chemistry) are
required. 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 aeries 153 of this subdivision indicates a
potential for human hazard. Residue data developed in accordance
with Subdivision O would then be required to obtain a temporary
tolerance.
(d) Toxicology data. The following data are required by 40
era § 158.165 to support an application for an experimental use
permit:
(1) Biochemical pest control agents not used on food crops:
(i) Acute oral toxicity (§ 152-10);
(ii) Acute dermal toxicity (§ 152-11);
(iii) Primary eye irritation (| 152-13);
(iv) Primary dermal irritation (§ 152-14); and
(v) Studies to detect gene mutation ($ 152-17).
(2) Biochemical pest control agents used on food crops:
(i) All studies listed in paragraph (d)(1) of this section; and
(ii) Cellular immune response studies (( 152-18).
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V.-l
Mijr^bial pest control a;-.".-,;: :>•-.'. .u>K-l .-.-. focv. cro;. s:
-• dermal i-festivit-.- (5 1?2-31);
{iii) Intravenous, intracerebral, intraperitoneal infectivity
v§ 152-33);
(iv) Primary dermal irritation (§ 152-34); and
(•/] Primary eye irritation {§ 152-35).
(4) Microbial pest control agents used on food crops:
(i) All studies listed in paragraph (d)(3) of this section;
(ii) Cellular immune response (§ 152-37); and
(iii} Tissue culture with viral agents (§ 152-39).
r. for ar. experir.ental -,;se perr.it, iaca from environ-T.-ental
fati ar.i egression studies according to section series 155 are
rsq^irei by 40 "??. I 155.155 fcr those biochemical and microbial
p*st ccr.trol ager.ts whose Tier I nor.target orcanisx test results
•frsr, section series 154) indicate that Tier II studies for environ-
rr.er.tal fite find expression should be conducted. For those pest
Jcntr;! aser.ts vhose Tier I r.ontarget organism test results indi-
cate no Tier II studies are necessary, no enviromental fate and
expression data are required for the application of a perr.it. In
those instances where field data from Tier II studies are required
ir. serti:- series 155 for a permit, any comparable or limited
fieli data would suffice in lieu of extensive field data; this
policy .3 needed to preclude development of extensive field data
without a permit in order to obtain information necessary to get
?«
a „,,,...; *
- «»<:...••_•.
Product performance data.
(1} General. In general, efficacy data will not be required
by 4D C??. § 152.165 to cuppa rt the issuance of an experimental use pernit.
t2) Except ions . (i) Initial pemits. Efficacy data may be
required, or: a case- by-case, for the following use categories:
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3 4
(A» Public K.*»alth -isos 1*ali.vi with ni-;r->icopic r^at oraan
.«:,d
(9) Use of cancelled or suspended pesticides.
(ii) Extensions, renewals, and amendments. Sunnaries of product
performance data collected under an experimental use permit may be
requested on a case-by-case basis by the Agency for purposes cf
•naicina:
(A) Determinations as to the need for additional quantities of
product requested by the applicant;
(BJ Evaluations of requests for permit extensions; and
(C) Assessments of requests for permit renewals.
Series 153: LABEL DEVELOPMENT
5 155-1 Product label requirements.
3ic.-ratior.al pesticides ar-s .;e.v»rally suhj«s.:t to .ill app'.icabl-.
labeling provisisns described in Subdivision a - Labeling Recuire-
ri.-.ts for Pesticides and Devices. Biochemical agents are vicjwe.i
essentially the same as conventional chemical pesticides with
respect to label requirements, but labeling for r.icrobial ag«::ts
iiffer principally with respect to th«2 ingredient statar.er.t. So:n«
instruct! an rsgar.linv ingredient statements for microbial ao-cr.t^
cars be derived fror. §§ 151-20 through -25 (Product Analysis) of
t'nis subiivision. Also, see § T5i-1{b)(6) regarding label clair-.s,
iirectior.s, precautions, and restrictions in rela-ion to use nitt^r
ir.fcrr.ati3.-. for biorational postirides.
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SEPORT DOCUMENTATION »-.
i»AGE
•.•• T -«o
I 3. *«.p
. 4. **!•« **• $«••'! •
Pesticide Assessment Guidelines
Subdivision Mr
. B to ra tio nal Pes tic id«
••••1 0»i t .._-
Hovember, 1982
O'«
! it.
Cuit
lit ">l| •« »».
TIONAL ro«u ;r: <4.i7i
tm«flr NTii-Jil
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