REGULATORY ASPECTS of MICROBIAL PEST CONTROL AGENTS (MPCA)
Zigfridas Vaituzis, Ph.D., Biopesticides and Pollution Prevention Division, EPA
Experimental Use Permit and Registration Data Requirements
An assessment of no unreasonable risk to the environment is performed based on testing data as required in 40 CFR Part 158.740(d), OPPTS 885 Series Guidelines1, PMRA Regulatory Directive DIR2001-022 and on other available information from the scientific literature. During the risk assessment process the registrants work closely with the regulatory agencies to develop procedures to mitigate a risk when one is identified in the data evaluation process. The Agencies follow a procedure by which the needs and concerns of agriculture, the biotechnology industry, the public and academia can be heard and discussed.
Non-target Organism and Environmental Expression data support assessments of the exposure and potential risk to non-target organisms and the fate of pesticides in the environment after use. Data requirements for non-target organism and environmental expression testing are tiered. Testing starts at Tier I, the lowest tier of these testing requirements. If the results of Tier I testing show adverse effects at field use rates, then testing at the next level may be required. Most MPCA's do not require testing beyond Tier I. If Tier I test results show adverse effects at the field use rates, consultation with the regulatory Agency is necessary on how to proceed with higher tier testing for experimental permits or registration. The goal of risk assessment is to assess the impact or, if necessary, restrict usage in habitats of wildlife at risk. Restrictive label language is usually sufficient to address the issues.
Data requirements vary depending on how a pesticide is used. The published environmental effects data requirement tables are divided into sections specifying what data are needed for broadly defined use patterns such as aquatic, terrestrial or greenhouse food or non-food uses. In general, outdoor use pesticides require more environmental data than indoor use products, because of increased potential exposure of non-target animals, plants, water and other environmental resources. Data requirements also differ for end-use products (EP) and manufacturing-use products (MP) which may be technical grade products or formulation intermediates. In general, all classes of data requirements apply to manufacturing-use products, and usually the required test substance is the technical grade active ingredient (TGAI).
Nontarget Organism Data Requirements
The information required to assess hazards to non-target organisms are derived from tests to determine pesticidal effects on birds, mammals, fish, terrestrial and aquatic invertebrates and plants. These tests include short-term acute, subacute, reproduction, simulated and/or actual field studies arranged in a tier system which progresses from the basic laboratory tests to the applied field tests. The test species are those expected to be exposed and can include indicator species such as bobwhite quail, mallard duck, sunfish, rainbow trout, Daphnia, honeybee, non-target insects and non-target plants.
In the sub-chronic toxicity/pathogenicity tests currently required, avian wildlife are exposed through either the diet or respiratory tract. The avian oral toxicity/pathogenicity study provides data on any direct toxic effects to avian wildlife following oral exposure to the microorganism or any toxins that may be produced during fermentation. This test would also provide data on pathogenic effects due to the microbial agent following oral exposure. The avian respiratory pathogenicity test provides information on the pathogenic effects of the active microbial ingredient on birds following exposure due to spray drift or direct spray application. The duration of both the avian oral and respiratory studies should be at least 30 days to permit time for incubation, infection, and manifestation of pathogenic effects in the test organism. In the instances where pathogenesis is suspected, attempts are made to isolate the causative organism to determine if it is the MPCA.
In both the avian oral toxicity/pathogenicity and respiratory pathogenicity tests, the test animals are evaluated by noting mortality, changes in behavior, pathogenic or toxic effects, gross necropsy, and histopathological examination including culture and isolation of the causal microbe from exposure sites, tissues or other organs showing anatomical or physiological abnormalities. In cases where cell or tissue preferences are known or suspected, those tissues are examined for gross anatomical or physiological changes. If no toxic or pathogenic effects are observed after exposure via oral and respiratory routes, then no further testing in birds is required. If effects are observed then Tier II, environmental expressions tests, would be required.
Data on wild mammal toxicity/pathogenicity are required on a case-by-case basis when data indicate that there is considerable variation in the sensitivity of different mammalian species to the effects of a microbial-based pesticide or where wild mammals would be expected to be exposed to a high dose under normal use. However, the toxicity/pathogenicity data in laboratory rodents submitted to evaluate hazards to humans are normally adequate to indicate potential hazards to wild mammals. Usually if no toxicity/pathogenicity effects are observed in these tests, no further testing of wild mammals would be required.
For microbial pesticides applied in terrestrial use patterns, where direct aquatic exposure is not anticipated, one freshwater fish and one freshwater aquatic invertebrate should be tested to assess toxicity and pathogenicity. These tests should be conducted as 30-day (for fish) or 21-day (for aquatic invertebrate) static renewal bioassays where the microbial inoculum is administered as a suspension in water, and also in the diet for fish, in the form of diseased host insects or treated feed. If any test animals die during the test, the cause of death (e.g., toxicity, pathogenicity) should be determined, if possible, and reisolation of the microorganism from the test organism's tissues should be attempted. Individual test animals should be removed periodically, if necessary, throughout the test period and at test termination for examination to assess pathogenicity. An important consideration in microbial pesticide aquatic studies is the need to keep the test substance in suspension and to measure the actual concentration of the test substance in the water column. The actual measured and the nominal concentration are usually different. The challenge of keeping as much material in suspension as possible is more difficult for the aquatic invertebrate Daphnia than for fish. Some laboratories have turned to stir bars and daphnid "shark cages" to protect the Daphnia from the agitation during testing.
Assessment of potential risk to non-target insects from the use of microorganisms which are insect pathogens is also an environmental concern. The non-target insect concern is evaluated by an examination of the published literature and toxicity/pathogenicity studies. The tier-testing scheme is based on a fairly extensive first tier which assesses toxicity and pathogenicity of the microbe to the honey bee and to a variety of species of predaceous, parasitic and other non-target insects of ecological significance. Selection of the predator/parasitic species should be representative of groups which will be exposed under the condition of proposed use and, if possible, which have some relationship to the target pest. Tier I testing also includes toxicity/pathogenicity testing with Daphnia, or, if available and appropriate, another aquatic insect species depending on use pattern. Data derived from the Tier I tests are used in conjunction with available information on use patterns, specificity of host range, fate, and other factors, to assess potential for adverse effects. If the test results indicate no adverse effects at maximum hazard doses, no further testing is required. By contrast, if toxicity or pathogenic effects are observed then Tier II testing, environmental expression, would be required. It should also be noted that the best routes of exposure in the Tier I tests will depend on the developmental stage and location of the non-target insect.
Similar concerns exist for assessing the potential hazards to non-target plants for microbial products based on plant pathogenesis.
The data and information obtained from the non-target organism and environmental expression tests described above allows for assessment of potential hazards from microbial pesticide exposure. However, in some cases data waivers may be appropriate for non-target testing requirements. Where non-target fish, plant, insect or bird exposure without toxicity and/or pathogenicity to the microbe in question can be documented in the scientific literature, a request to waive the data requirement may be entertained. Consequently, non-target pathogenicity testing may not be necessary if the microbial pesticide's natural environmental distribution includes the habitat of the non-target organism species normally tested in Tier I pathogenicity studies, and the microbial pesticide has never been found in association with non-target organism infectivity and disease. Waivers may also be justified if there is a reasonable argument that the non-target organisms for that particular environment will not be exposed to the microbial pesticide. Acute toxicity studies, however, may be required where the agricultural doses would greatly exceed the amount of the microbe and its metabolites naturally found in the environment.
Tiered Testing
It is desirable to have a high level of confidence that no unreasonable adverse environmental effects will result from actual use of MPCAs. Toward this end, the guidelines in Tier I reflect a maximum hazard approach to testing. The Tier I test requirements represent a reasonable approach to evaluating risk related to the use of biopesticides, and is one in which negative results would allow a high degree of confidence in that no unreasonable adverse effects are likely to occur from the actual use of MPCAs. If adverse effects are observed at maximum doses, then sequentially lower doses should be tested to determine an LD50, LC50 or ID50. Where Tier I testing shows a hazard at doses approaching the expected environmental concentrations, Tier II or higher tier testing (to assess population level effects) would be required. In addition, definitive Tier II data showing that the MPCA will not survive or persist in the environment to which it is applied, can be submitted as support for a request for waiver of some or all of Tier I testing requirements.
Non-Target Organism (NTO) Data Requirements: The following testing is required in order to assess hazard of microbial pest control agents to non-target organisms.
| Tier I: | avian oral avian pulmonary/inhalation/injection wild mammals freshwater fish estuarine/marine fish terrestrial arthropods |
aquatic arthropods non-arthropod invertebrates non-target microorganisms terrestrial non-target plants aquatic non-target plants |
If adverse effects are observed in Tier I at maximum doses, then sequentially lower doses should be tested at to determine an LD50, LC50 or ID50; Tier II testing is also triggered. |
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| Tier II: | Terrestrial, freshwater and marine or estuarine environmental expression testing. (environmental fate).
Potential environmental persistence and exposure to the MPCA is estimated by means of terrestrial, freshwater and marine or estuarine environmental expression testing taking into account the application rates, movement in soils and population dynamics. |
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| Tier III: | Chronic, reproduction, life cycle and population effects (host range) testing
If expression tests show significant exposure potential to Tier I susceptible species, additional studies are required on these species to examine chronic, reproduction, life cycle and population effects. |
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| Tier IV: | Simulated (microcosm) or actual field testing
Simulated or actual environmental field tests are designed on a case-by-case basis to evaluate any specific environmental problem that cannot be resolved by lower tier testing |
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If the results from Tier II tests show that the MPCA persists or survives in the environment at significant levels, Tier III studies are designed to show effects of chronic exposure to these levels on fish and wildlife. If it is indicated that there may still be a problem, Tier IV studies (simulated or actual field studies) may be able to determine if there is an undesirable effect under actual use conditions. |
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Data waiver requests:
If the MPCA belongs to a group of microorganisms that never have been found associated with any disease, a case may be made for reducing, or waiving, the testing requirements. Definitive ecological exposure data showing that the biopesticide will not survive or persist in the environment should be good support for a request for waiver of some or all of Tier I testing requirements. The data requirements will not always be appropriate for every product. Some products have biological properties or use patterns which would make particular data requirements inappropriate because it would not be possible to generate the data or because the data would not be useful in evaluation of risk. The data requirements that are inappropriate will be waived on a case-by-case basis in response to written requests. In general, however, the written waiver requests have to address and satisfy each non-target data requirement with information other than the required testing.
Principles for justifying data waivers.
| (i) | The full battery of tests for registration of MPCAs was designed to give basic hazard and exposure information for a microorganism with totally unknown properties. In actual practice, an MPCA is usually well identified, which may facilitate prediction of its properties and behavior. This is particularly true for the areas animal, insect and plant pathogenicity. Veterinary and agricultural scientists have identified most microorganisms associated with diseases. If an MPCA is taxonomically similar to a clinically or agriculturally significant microorganism, this particular area of concern should be examined closely, possibly by requiring additional testing beyond that specified in the guidelines. Conversely, if the MPCA belongs to a group of microorganisms that have never been found in association with any disease, a case may be made for reducing, or waiving, the testing requirements for this area of concern. |
| (ii) | The regulatory Agencies' guidelines contain provisions for granting waivers for data requirements in response to specific written requests by applicants. The applicants are encourages to discuss their preliminary testing plans with the regulatory Agency scientists. Waivers of some testing requirements may be appropriate for certain MPCAs. This tailoring of the testing battery on a case-by-case basis relies on both an accurate description of the MPCA and the existence of a reliable taxonomy for the class of microorganism to which it belongs. Some microorganisms have been more closely examined than others and have a larger data base from which to draw conclusions. In addition, certain kinds of microorganisms are more amenable to classification than others. In general, animal and plant pathogenic bacteria have been best classified due to their health and economic significance. Other microorganisms, particularly protozoa and fungi, might not be as well studied or described, and it may be difficult to predict their properties reliably from a taxonomic description. In this case, it may be more difficult to justify waiving test requirements. |
| (iii) | An additional factor in determining the extent of testing that may be necessary for risk assessment is the degree of species specificity shown by the MPCA. This is of primary importance in assessing ecological risk. Most MPCAs are designed to produce adverse effects against a target species. Careful scientific consideration on a case-by-case basis must be given to the selection of non-target species to be tested (e.g., beneficial and environmentally or commercially significant insects, plants, or wildlife) in order to include species that are most likely to be susceptible. |
| (iv) | Because of the difficulty in providing definitive exposure predictions from currently available ecological methods, hazard testing on non-target organisms should be submitted initially (Tier I tests). If significant adverse effects are identified in Tier I tests, ecological exposure tests (Tier II) are performed to attempt to quantify levels of the MPCA to which the susceptible non-target species may be exposed. Although normally requested at a Tier II level, definitive ecological exposure data showing that the MPCA will not survive or persist in the environment would be good support for a request for waiver of some or all of Tier I testing requirements. |
Candidate Test Species
Selection of Nontarget Test Species
Consideration should be given to the testing of representative or indicator species from some or all of the 7 broad NTO groups (listed in Tier I) and then species representative of the geographic region (ecozone in Canada) or ecosystem where the MPCA is to be applied. These should be species that are generally found across North America, be of some environmental or economic importance, or be a common (indicator) test species for assessing the effects of a variety of environmental stressors. The selected candidates should be reasonably representative, readily available and able to be reared in a laboratory setting.
Age of Test Organisms
It is highly desirable to test immature birds, mammals and fish which have not yet developed immunity to a large number of microbes. Immature animals are potentially more susceptible to infection and possibly to effects of any toxin(s) produced by the MPCAs. Arthropods, non-arthropod invertebrates and plants should be treated either at the time (life-stage) of most likely exposure in the field or at the time of most likely susceptibility to the MPCA.
Avian test species
The guidelines provide that young bobwhite quail or mallard ducks be tested in Tier I tests. The upland game species (bobwhite quail) is preferred for terrestrial uses, while the waterfowl (mallard duck) should be tested where considerable aquatic exposure is anticipated. Birds between 14 and 28 days of age at the beginning of the test period should be used in the avian oral toxicity/pathogenicity testing.
Wild mammals
Need for testing dependent on results of Tier I toxicity/infectivity tests on laboratory animals for human health risk assessments.
Aquatic wildlife
Considerable judgment will be required to properly employ site of application as a criterion for species selection. While many uses obviously entail direct application to water (e.g. mosquito control and aquatic weed control), less obvious or borderline uses will also be considered aquatic uses. Some examples that fall into the latter category are applications to forests, drainage ditches, riverbanks, and partially aquatic crops such as rice. Widespread applications to major crops such as cotton, soybeans, and corn could also warrant expanded testing if these crops are grown near bodies of water. To the extent possible, the regulatory Agencies will rely on their experience with the classical chemical pesticides in distinguishing between terrestrial and aquatic use patterns in borderline situations.
Fish species
The guidelines provide that the species tested be selected from the recommended list with the exception of goldfish (warmwater species – bluegill sunfish, channel catfish, and fathead minnow; coldwater species – rainbow trout, brook trout, coho salmon). These species are desirable test organisms for several important reasons: They are used to evaluate chemical pesticides, and the regulatory Agencies have considerable background data on these species; standard methods for the care and handling of these species are available; and the species are widely distributed, are generally available, and have a variety of food habits and habitat requirements.
Consideration should be given to testing species representative of the geographic region or ecosystem where the MPCA is to be applied. Species likely to prey upon or scavenge the diseased target host animals should be tested when appropriate.
Unless there are other overriding considerations, the rainbow trout should be used as the freshwater fish test species for terrestrial uses with no direct aquatic exposure. (It is a desirable test animal because it is partially insectivorous). Two freshwater species – coldwater (trout) and warmwater (bluegill sunfish, channel catfish, fathead minnow) for uses with direct aquatic exposure (e.g., forestry, drainage ditches, mosquito/black fly control). The sheepshead minnow for estuarine/marine species if significant exposure is expected and the MPCA can tolerate salinity 10 ‰. The recommended routes of exposure are suspension in the test water (aqueous exposure) and/or dietary, in the form of diseased target host or incorporation of MPCA into standard feed.
Aquatic Arthropods
Tier I testing, will include toxicity/pathogenicity testing with Daphnia, or a species of aquatic insect, or both, depending on use pattern. Detection of pathogenicity/toxicity in Tier I testing will automatically lead to expanded testing which, if the impacted site is fresh water, will most likely involve testing with aquatic insects. Test species can include representatives of up to 14 freshwater and 6 estuarine taxa, but usually the freshwater daphnid (Daphnia magna) and marine shrimp (Paleomonetes vulgaris).
Due to the broad phylogenetic spectrum from which the investigator may choose, it is difficult to select the most appropriate aquatic invertebrate. Generally, a test organism that is phylogenetically closest to the target host should be chosen. Such a test organism would be the most likely to be susceptible to infection by the MPCA. It would be appropriate to choose an aquatic insect (e.g., caddisfly) as the nontarget aquatic invertebrate test species when evaluating a MPCA whose target host is an insect.
Daphnia, a Cladoceran, has the advantage of having considerable background data for comparative purposes. In addition, Daphnia is useful to demonstrate a bioconcentration effect. This is due to the filter feeding habits of Daphnia and is a desirable feature in terms of assuring that the test animal ingests the microorganism. Both Daphnia and certain other aquatic insects have the advantage of a short life cycle or aquatic phase, and both undergo periods of natural stress and potential susceptibility to the microorganism as a consequence of molting.
Two methods of pesticide administration should be considered:
| (i) | Suspension in the test water (aqueous exposure); and, |
| (ii) | Dietary, in the form of diseased target host animals or incorporation of the MPCA into a standard feed. |
Terrestrial Arthropods
Approach. Assessment of potential non-target arthropod hazard from uses of MPCAS is made difficult by a number of factors:
| (i) | Most MPCAs will be specifically selected and/or designed for their ability to control pest insects. Nontarget insects are the organism group most at risk, being relatively closely related to the target organism in most cases. |
| (ii) | While there are few nontarget insects that have been shown to be economically important to humans, there are many nontarget insects which have an important role in ecological processes and may benefit humans indirectly. |
| (iii) | Unlike chemical pesticides, many microbials will exert their effect through pathogenicity as well as toxicity. Adequate assessment of pathogenicity requires time to evaluate the MPCA for infectivity and for its ability to reproduce or develop in the test insect. |
| (iv) | The host range is an important factor in hazard evaluation for an MPCA. A problem here is that extrapolation, even across species lines, is often not dependable. For this reason, the guidelines provide for testing with representatives from a number of "beneficial insect" taxa. Information from these tests is used in conjunction with host range data (developed during efficacy testing) to develop a clearer idea of the overall insect host range. |
| (v) | Tier I testing may be more extensive in some cases than the baseline data requirements. However, there should be very few microbials which require effects testing beyond the Tier I level. |
| (vi) | The tier-testing scheme for MPCAs is based on a fairly extensive first tier. The purpose of the Tier I testing is to assess toxicity and pathogenicity of the MPCA to the honey bee and to three species of predaceous and parasitic insects. Selection of the predator/parasite species to be tested should take into account such factors as the likelihood of exposure to the MPCA, phylogenetic proximity of the test species to target pest species, and similar relationships. A rationale for selection should be developed by the registrant. |
Insect test species
| (i) | Testing should be performed on a major pollinator and three other species of insects, representing at least two of the following groups – parasitic dipterans, predaceous hemipterans, predaceous coleopterans, predaceous mites, predaceous neuropterans, parasitic hymenopterans. |
| (ii) | Selection of test species is the responsibility of the registrant in consultation with the regulatory agencies. Rationale for selection must be provided. |
Test species can include representatives of up to 24 taxa, but usually (for non-insecticidal MPCAs) include any beneficial indicator taxa:
Indicator taxa:
honey bee (adult and/or larvae)
Plus 3 species from at least 2 groups:
parasitic dipterans
predaceous hemipterans
predaceous coleopterans
predaceous mites
predaceous neuropterans
parasitic hymenopterans
Route of Exposure: topically and/or dietary
Non-Arthropod Invertebrates
The need for testing is based on the expected exposure if the MPCA is related to a known pathogen or is intended to control invertebrates. If required, susceptible host(s) and/or environmentally/economically important species are tested, such as the earthworm (for terrestrial exposures) and a mollusc (for aquatic exposures).
Microorganisms
Microorganisms are exempt from testing unless there is reason to expect potential for adverse effects on environmentally/economically important microbial species and/or microbial processes. If testing is required, testing of major biogeochemical processes, e.g., CO2 evolution, nitrogen transformation, cellulose degradation, etc. will be required.
Plants
The number and type of species tested is dependent on whether the MPCA is herbicidal or non-herbicidal and whether the MPCA is intended for terrestrial or aquatic use. MPCAs that do not resemble any known plant pathogen may require little, if any, plant testing. Observations of phytotoxicity/phytopathogenicity from efficacy trials (laboratory and field) can be used as supportive data for MPCAs not related to known or suspected plant pathogens.
For MPCAs likely to cause plant toxicity/pathogenicity up to13 terrestrial families with focus on commercial crops may have to be tested:
| Apiaceae (Umbelliferae)
Asteraceae (Compositae) Brassicaceae (Cruciferae) Chenopodiaceae Cucurbitaceae Fabaceae (Leguminosae) Liliaceae |
Malvaceae
Poaceae (Gramineae) Polygonaceae Rosaceae Solanaceae Pinaceae (forestry uses)
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Up to 6 aquatic vascular plant families:
Lemnaceae
Potomogetonaceae
Haloragaceae
Typhaceae
Cyperaceae
Alismaceae
Additional testing may be required on at least one of three species each of:
Chlorophyceae (green)
Cyanophyceae (blue-green)
Bacillariophyceae (diatoms)
Test substance
Microorganisms used as pesticides could be applied in any one of a combination of naturally existing forms. It is preferable that the test organism be exposed to the most infectious form whenever infectivity is the primary hazard of concern. Similarly, when toxicity (e.g., a microbial toxin) is the hazard of concern, the test organism should be exposed to a form of the MPCA in which the toxin would be produced in the greatest amount and most readily available. Unfortunately, there is no easy way to determine which is the most infectious or toxic form of the 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, the active vegetative cells of a bacterium, or the infectious hemolymph may be more appropriate than vegetative cells or polyhedryda, respectively.
Test substance for environmental studies
It is advised that appropriate representatives of the regulatory Agencies be consulted prior to testing in order to determine the form/purity of the MPCA that should be tested to support the registration of each manufacturing-use product (MP) and each end-use product (EP). It is recognized that certain forms of the MPCA may be inappropriate for certain tests. In general, the form (e.g., vegetative cell, spore, cyst, virion) of the microorganism to be tested should be equivalent to the form that is intended for registration. The test microorganism also should be equivalent to that intended for registration with respect to stage of growth, possession of organelles and appendages, and expression of phenotypic traits . If significant exposure to other forms of the microorganism is expected, or if changes in form of the microorganism occur, or are expected to occur in target, or non-target species, these forms may have to be tested also. In general, the following principles should be followed: Aquatic test substance
The substance to be tested will depend in part on the method of pesticide administration used in the study. It is essential to test the most challenging form of the microorganism (in terms of pathogenicity or toxicity). It is equally important to test the form of the microorganism to which non-target aquatic animals are most likely to be exposed. These objectives should be achievable, provided it is known which form is most challenging and which form is most likely to be encountered by the non-target animal. The technical grade of the active ingredient should be used for all exposures. The formulated product should be tested if it is to be applied directly to water.
| (i) | Tests requiring use of the technical grade active ingredient (TGAI) shall be conducted with the manufacturing-use product if the TGAI and MP are identical, or with the TGAI used to produce the manufacturing-use or end-use formulated pesticide product if not identical. |
| (ii) | The lot of the substance tested should be the same throughout the duration of the study, and the test sample should be stored under conditions that maintain purity and stability. If the stability of the test substance cannot be maintained for the duration of the study or if, for other reasons, it is not possible to use the same lot throughout the test, subsequent lots of the test substance shall be selected to be as nearly identical to the original lot as practical. Chemical or biological assays shall be performed to ensure composition identity and consistency. |
| (iii) | Each lot of the test substance shall be analyzed, to the limits of technical feasibility, and the name and quantities of ingredients, contaminants, and impurities listed. The determination shall include the quantity of unknown material, if any, so that 100 percent of the test sample is accounted for. |
| (iv) | If the test or control substance is to be incorporated into feed or other vehicle, the period during which the test or control substance is stable or viable in such a mixture should be determined prior to the start of the study. No mixture of test or control substance with the feed or vehicle shall be maintained or used during a period exceeding the known stability or viability of the test or control substance in the mixture. Alternatively, determinations of the stability or viability of the test or control substance in random samples of the diet or vehicle mixture shall be made at least monthly during the study to ensure that proper mixing, formulation, and storage procedures are being followed and that the appropriate concentration of the test or control substance is 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 shall be determined prior to the start of the study and, each time a new mixture is prepared. Random samples of the mixture shall be analyzed at least monthly to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. |
| (vi) | In addition to or in lieu of data otherwise required by the regulatory guidelines, after consultation with the applicant, it may be necessary to derive data from testing conducted with: |
| (vii) | In addition to or in lieu of data otherwise required by the regulatory guidelines, after consultation with the applicant, it may be necessary to derive data from testing conducted with: |
| (A) | An analytically or microbiologically (e.g., PIB for viruses) pure grade of an active ingredient. |
| (B) | The labile form of infectious material (e.g. non-occluded virus). |
| (C) | An inert ingredient of a pesticide formulation. |
| (D) | A contaminant or impurity. |
| (E) | A metabolite (from animals or plants) or degradation product of an active or inert ingredient. |
| (F) | The end-use formulated product. |
| (G) | Any additional substance (including other pesticides recommended for tank mixing with the test substance) that enhances the virulence or toxicity of the product for which registration is sought. |
| (H) | Any combination of the substances mentioned above. |
Administration of test substance and vehicles
| (i) | The manner of administration or application of the test and control substance for environmental testing shall be selected to maintain accuracy of the dosage or treatment. |
| (ii) | No part of the test system shall contain added antibiotic substances. |
| (iii) | A vehicle other than water or saline used to dissolve or dilute the test substance or positive control substance shall be chosen to possess the following characteristics if possible: |
| (A) | It does not alter the absorption, distribution, metabolism, or retention of the test substance. |
| (B) | It does not alter the chemical or biological properties of the test substance or enhance, reduce, or alter the pathogenic or toxic characteristics of the test substance. |
| (C) | At the levels used in the study, it does not produce physiological effects and is nontoxic. |
| (D) | It should be identical to, or closely resemble the vehicle, if any, used in the pesticide product. It should be identical to the vehicle if possible. |
Insect test substance
Any substances used to enhance virulence should be tested along with the test substance.
Maximum hazard dosage levels
Unlike environmental levels of chemical pesticides, which generally decrease following application, the environmental levels of MPCAs and any associated toxins may, at least temporarily, increase when the product is effective. Therefore, the maximum hazard dose for Tier I testing will be based on some safety factor times the maximum amount of active ingredient (MPCA or its toxin) expected to be available to terrestrial and aquatic plants and animals in the environment. The target hosts (e.g. insects) are likely to contain the highest concentration of the MPCA that will be available to non-target terrestrial wildlife and aquatic animals following a pesticide application.
In most cases, testing at one maximum hazard dosage level is expected to be sufficient to evaluate effects for these MPCAs. MPCAs that are toxin-producing are more likely to produce a log-probit response. In most cases multiple groups would be necessary in order to quantify the hazard of these organisms. If there are no effects at the maximum hazard dose, low doses will not be necessary.
In Tier I, test organisms should be exposed to a maximum hazard/maximum challenge concentration (MCC) of the MPCA.
For avian toxicity testing, MCC is a function of some safety factor that is based in part on the route of administration and the MPCA concentration in the TGAI: - Oral = 5.0 mL/kg bw × Weight of bird (kg) - Pulmonary = 0.2 mL/kg bw × Weight of bird (kg) Intravenous = 0.5 mL/kg bw × Weight of bird (kg) Intraperitoneal = 2.0 mL/kg bw × Weight of bird (kg) For aquatic NTOs (fish, invertebrates, plants): - 106 viable units of MPCA per mL of water; or - 1000X expected environmental concentration (EEC) of MPCA, immediately following a direct application at the maximum label rate to a 6-inch (15-cm) layer of water, whichever is greater or achievable (water quality considerations). - for artificial dietary exposures, MCC should be equivalent to the maximum concentration found in the target; or feed diet of maximally infected target For terrestrial NTOs (insects, invertebrates, microorganisms): - 106 active units of the MPCA per gram of soil; or - 1000 times the expected environmental concentration of the MPCA, immediately following a direct application at the maximum label rate to a 15-cm layer of soil, whichever is greater or achievable - for topical exposure tests, exposure to a concentration of the MPCA that is equivalent to 100X the maximum application rate - for artificial dietary exposures, MCC should be equivalent to the maximum concentration found in the target; or feed diet of maximally infected target
Whenever feasible, dosage shall be in suitable increments up to 100 x the LD50 or LC50 of the pathogen in its natural host, or 10-100 x the recommended field dosage. In cases where it is difficult to determine the maximum concentration in the target, feed a diet treated with an application of the MPCA equivalent to 100X the maximum label rate
Maximum hazard routes of administration.
Various routes of administration (dosing) are provided for in the regulatory Agency guidelines and are chosen to reflect ``natural'' exposure routes. These routes – oral and respiratory for birds, aquatic and food exposure for aquatic organisms, and the oral route for insects – can best define the hazard to non-target organisms in the wild.
Citations:
1 885 Microbial Pesticide Test Guidelines
2 Pro98-01 Guidelines for the Registration of Microbial Pest Control Agents and Products