Microbial & Biochemical - REBECA

Microbial & Biochemical 

US Environmental Protection Agency 

US Environmental Protection Agency 

Office of Pesticide Programs 

Office of Pesticide Programs 

Biopesticides & Pollution Prevention Division 

Biopesticides & Pollution Prevention Division 

William R. Schneider, Ph.D. 

William R. Schneider, Ph.D. 

schneider.william @ epa.gov 

schneider.william 703-308-8683@ epa.gov 

703-308-8683 

Pesticide Data Requirements

Microbial & Biochemical 

Pesticides 

• The first microbial pesticide was 

registered in 1948 

• Mid 1970’s – began development of 

data requirements 

• Microbial Pesticides needed different 

kinds of tests 

• Some naturally-occurring chemical 

pesticides could be evaluated with a 

reduced set of data requirements. 

1

History 

2 

• NSF report - 1977 

– Biological Substitutes for Chemical Pesticides, 

• Proposed Data requirements – all Pesticides 

– Published FR July 10, 1978 (40 CFR 163) 

– Biocontrol organisms – case by case data 

• Policy statement: Regulation of Biorationals 

– Published FR May 14, 1979 

• American Institute of Biological Sciences - 1979 

– Human Hazard Evaluation Scheme for Biorationals 

• Subdivision M guidelines 1982, NTIS 

• Data Requirements – all Pesticides 

– 40 CFR 158 (FR October 24, 1984) (1983 SAP)

History (people) 

3 

• Martin Rogoff 

• Reto Engler 

• Fred Betz 

• Al Vaughan 

• Ron Ney 

• Bob Hitch 

• Stuart Cohen 

• Bill Preston 

• Ed Gray 

• Amy Rispin 

• Bill Schneider 

• Roy Sjoblad 

• Tom McClintock 

• Janet Andersen 

• Debby Edwards 

• Morris Levin 

• Zig Vaituzsis 

• Doug Urban 

• Bob Pilsucki 

• Bill Hazel 

• John Couch (ORD) 

• Clint Kawanishi (ORD) 

• Anne Fairbrother (ORD) 

• Fred Genthner (ORD) 

• Clay Beegle 

• John Kough 

• David Bays 

• Bob Rose 

• Phil Hutton 

• Willie Nelson

Microbial Pesticides: The Past 20 years 

• Pre-1984: 17 microbial 

pesticides were 

registered 

• Post- 1984: Over 70 

microbial pesticides 

registered 

60 

50 

40 

30 

20 

10 

0 

Pre-1984 

Post- 

1984 

4

History 

• Subdivision M guidelines revision, NTIS 

1989 (1988 SAP) 

• Draft 40 CFR 158 Data Requirements 

– Corresponded to the revised guidelines 

– Presented to, and approved by the SAP 1994 

– Not published pending revision of other 158 

sections 

• Harmonized Guideline revisions 1995 

– published on internet 

– Some still being revised 

5

Scope of this Proposal 

6 

• Clarify definitions 

– biochemical (40 CFR Part 158) 

– microbial pesticides (40 CFR Parts 158 and 172) 

• Update data requirements for biochemical and 


• Presentation format: 

– Discuss Microbials & then Biochemicals 

• Definitions described first 

• Data requirement changes described second 

–1 st slide will show original 1982 data requirements 

– The following slide(s) will present changes in red text 

– Deleted data requirements will be crossed out 

– Test name changes will appear in italics

Data Requirements Revisions 

7 

• Current tables reference 1982 guidelines 

– changed to harmonized guideline (numbers & names) 

• Some studies are no longer needed 

– Some harmonized guidelines were never written 

• Because we don’t expect to need them 

• The tests wouldn’t give answers we could use 

– We currently have to ask for waivers for each one 

• Some new studies have been added 

– To provide better options to evaluate positive results seen at lower 

tiers. 

– To harmonize with conventional pesticides 

• Test substances (TGAI, MP, EP) have been changed for 

some tests 

• Test notes have been revised to give better guidance.

Microbial Pesticides 

definition 

8 

• Original: 

– Microbial pesticides include microbial entities such as 

bacteria, fungi, viruses, and protozoans. 

• Proposed: 

– Microbial pesticide is a microorganism intended for 

preventing, destroying, repelling, or mitigating any 

pest, or intended for use as a plant regulator, defoliant, 

or dessicant, that: 

(1) Is a eucaryotic microorganism including, but not 

limited to, protozoa, algae, and fungi; 

(2) Is a procaryotic microorganism, including, but not 

limited to, bacteria; or 

(3) Is an autonomous replicating microscopic element, 

including but not limited to, viruses.

Microbial Pesticides 

78 registered 

9 

• Bacteria (39) 

– 17 Bacillus thuringiensis subspecies 

– 10 other Bacillus species 

– 10 Pseudomonas species 

– 2 Agrobacterium isolates 

• Fungi (29) 

• Baculoviruses (7) 

•Yeast(2) 

• Protozoa (1 )

Risk Assessment 

10 

• Hazard 

– Toxicity, Pathogenicity 

• Exposure 

– Biopesticide aspects 

• Scale of use, use patterns, application rates 

• Persistence, degradation, mobility 

• Microbials: Population dynamics, infectivity, residues 

– Used less for microbial pesticides 

• Difficult analysis for microbials that multiply in the 

environment 

• Microbial toxins may need analysis 

– Biochemical pesticides occur naturally in the 

environment 

• Exposure data required only if toxicity concerns are identified 

• Risk 

– Non-targets, humans, domestic animals, endangered 

species

Microbial Data Requirements 

(original) 

• Product Analysis 

– Product Identity*** 

– Manufacturing Process 

– Discussion of unintended ingredients 

– Analysis of samples 

– Certification of limits 

– Analytical methods 

– Physical & Chemical Properties 

– Submittal of samples 

11


(proposed) 


– Produc Identity*** 





– Analytical methods (moved to Product ID) 

– Physical & Chemical Properties (itemized) 

– Submittal of samples (to culture collection) 

12


(original) 

• Human Health Tier 1 Studies 

– Oral toxicity/pathogenicity - 3 dose levels 

– Dermal tox/path 

– Pulmonary tox/path 

– Iv, ip, ic tox/path 

– Immune response 

– Tissue culture – for viruses 

– Acute studies on formulated product 

– Toxicity: oral, inhalation, dermal 

– Irritation: eye, dermal (may include toxicity) 

– Hypersensitivity study 

– Hypersensitivity reporting 

13


(proposed) 

• Human Health Tier 1 Studies 

– Oral toxicity/pathogenicity – Maximum Hazard Dose 

– Dermal tox/path – (replaced by Dermal toxicity) 

– Pulmonary tox/path 

– Iv, ip, ic tox/path – ic exposure not practical 

– Immune response - not relevant for microbials 

– Cell culture – for viruses 

– Acute studies on formulated product 

– Toxicity: oral, inhalation, dermal 

– Irritation: eye, dermal (if irritation seen in dermal tox study) 

– Hypersensitivity study (not relevant for microbials) 

– Hypersensitivity reporting 

14


If effects seen in Tier I Human Health (original) 

• Tier II Studies 

– Acute toxicity oral & inhalation (viruses & protozoa) 

– Subchronic oral (protozoa) 

– Residue studies 

– Ip/ic tox path (2 other species, 4/6 months duration) 

– Primary eye/dermal (dose: the use dilution of EP) 

– Immune response (no protocols in old guidelines) 

– Teratogenicity (for viruses that damage cell cultures) 

– Virulence Enhancement (LD50 after serial passage) 

– Mammalian mutagenicity (dosing: microorganisms) 

15


If effects seen in Tier I Human Health (proposed) 

• Tier II Studies 

– Acute toxicity (evaluate any toxicity seen in Tier I) 

– Subchronic (evaluate any persistence without toxicity) 

– Residue studies (for mammalian toxic components) 

– Ip/ic tox path (2 other species, 4/6 months duration) 

– Primary eye/dermal (dose: the use dilution of EP) 

– Immune response (no protocols in old guidelines) 

– Teratogenicity (for viruses that damage cell cultures) 

– Virulence Enhancement (LD50 after serial passage) 

– Mammalian mutagenicity (dosing: microorganisms) 

16


If effects seen in Tier II Human Health (original) 

• Tier III Studies 

– Oncogenicity 

– Teratogenicity 

– Mutagenicity 

– Chronic feeding 

17


If effects seen in Tier II Human Health (proposed) 

• Tier III Studies 

– Carcinogenicity (conventional chemical guidelines) 

– Reproductive fertility effects 

– Mutagenicity (not useful for testing living microbials) 

– Chronic feeding (not relevant for living organisms) 

– Immunotoxicity (added for viruses if needed) 

– Infectivity/Pathogenicity analysis (added for pathogens) 

18


Non-target Organisms & Environmental Fate (original) 

• Tier I Studies 

– Avian Oral (quail or mallards) 

– Avian Injection (quail or mallards) 

– Wild Mammal testing (rarely needed) 

– Freshwater Fish testing 

– Freshwater Aquatic Invertebrate testing 

– Estuarine & Marine Animal testing 

– Nontarget Plant studies 

– Nontarget insect testing 

– Honeybee testing 

19 

• Tier II studies - environmental expression tests 

– Terrestrial, Freshwater, and Marine or estuarine



• Tier I Studies 

– Avian Oral (option of passerine species) 

– Avian Inhalation (rarely needed) 

– Wild Mammal testing (rarely needed) 

– Freshwater Fish testing (only if exposed) 

– Freshwater Aquatic Invertebrate testing ( " ) 

– Estuarine & Marine Fish & Invertebrates 

– Nontarget Plant studies (if similar to plant pathogen) 

– Nontarget insect testing (only insecticides) 

– Honeybee testing (guidelines now include larvae) 

20 

• Tier II studies – ( no changes ) 

– Terrestrial, Freshwater, and Marine or estuarine



• Tier III Studies (mesocosm testing) 

– Avian pathogenicity/reproduction test 

– Special aquatic tests (reserved, no protocols) 

– Nontarget plant studies (case by case protocols) 

– Terrestrial wildlife and aquatic organism testing 

– Definitive aquatic animal tests (fish & invertebrates) 

– Aquatic embryo larvae and life cycle studies (↑) 

21


Non-target Organisms & Environmental Fate (proposed) 

• Tier III Studies (mesocosm testing) 

– Avian chronic pathogenicity/reproduction test 

– Special aquatic tests (reserved, no protocols) 

– Nontarget plant studies (case by case protocols) 

(plant study now at Tier IV, field testing) 

– Terrestrial wildlife and aquatic organism testing 

– Definitive aquatic animal tests (fish & invertebrates) 

22 

– Aquatic embryo larvae and life cycle studies (↑) 

Redesigned the above 3 studies with new protocols: 

– Aquatic invertebrate range testing 

– Fish life cycle studies 

– Aquatic ecosystem test (multiple species)



• Tier IV Studies (field testing) 

– Simulated and actual field tests (birds, mammals) 

– Simulated and actual field tests (aquatic organisms) 

– Simulated and actual field tests (insect predators, 

parasites) (reserved) 

– Simulated and actual field tests (insect pollinators) 

(reserved) 

23


Non-target Organisms & Environmental Fate (proposed) 

• Tier IV Studies (field testing) 

– Simulated and actual field tests (birds, mammals) 

– Simulated and actual field tests (aquatic organisms) 

– Simulated and actual field tests (insect predators, parasites) (reserved) 

– Simulated and actual field tests (insect pollinators) (reserved) 

None of the above had protocols - replaced by field test 

guidelines, below, for conventional chemical pesticides: 

– Field testing for terrestrial wildlife 

– Field testing for aquatic organisms 

– Simulated or actual field tests: 

– (birds, mammals) (aquatic organisms) 

– (insect predators, parasites) (insect pollinators) 

24 

–(plants)

25 

Summary: Microbial Pesticides 

New data requirements (at Tier III): 

– Infectivity/pathogenicity analysis and immunotoxicity (viruses) 

Revised data requirements: 

– Test notes better describe when studies are needed 

– Listed studies required for Physical/chemical properties and Residues 

– Analytical methods moved: Product analysis to Product Identification 

– Samples to be submitted to a culture collection instead of Repository 

– Dermal tox/pathogenicity study replaced by acute dermal toxicity 

– Avian injection test replaced by an avian inhalation test 

– Tier III plant study revised to a Tier IV field test. 

– 3 Tier III aquatic organism tests revised with new protocols 

– All Tier IV non-target field tests replaced by chemical pesticide field 

tests 

Deleted data requirements: 

– Hypersensitivity studies, i.c. tox/path, Immune response; 

Mutagenicity, Teratogenicity, Virulence enhancement, Chronic 

feeding, and Tier II ip/ic tox/path, and Primary eye & dermal

Biochemical Pesticides 

Definition - original 

26 

• Biochemical and microbial pesticides are 

generally distinguished from conventional 

chemical pesticides by their unique modes of 

action, low use volume, target species 

specificity or natural occurrence. 

• Biochemical pesticides include, but are 

not limited to, products such as 

semichemicals (e.g. insect pheromones), 

hormones (e.g. insect juvenile growth 

hormones), natural plant and insect 

regulators, and enzymes.


Definition - proposed 

27 

• (1) Is a naturally-occurring substance or 

structurally-similar and functionally identical 

to a naturally-occurring substance; 

• (2) has a history of exposure to humans and 

the environment demonstrating minimal 

toxicity, or in the case of a syntheticallyderived 

biochemical pesticide, is equivalent to 

a naturally-occurring substance that has such a 

history; and 

• (3) Has a non-toxic mode of action to the 

target pest(s).


• include but are not limited to: 

– (1) Semiochemicals (Insect pheromones 

and kairomones), 

– (2) Natural plant and insect regulators 

– (3) Naturally-occurring repellents and 

attractants, and 

–(4) Enzymes 

28

Non-Toxic Modes of Action 

examples 

• Lures/Attractants/Repellents (Irritants) 

• Suffocation 

• Dessication 

• Coatings 

• Systemic Acquired Resistance induction 

29 

• Growth/developmental changes (IGRs, PGRs)


160 registered 

30 

Semiochemicals (Pheromones) (50) 

• (Z)-9-Tricosene (muscalure) attracts house flies 

Insect growth regulators (4) 

• Azadirachtin – insect growth regulator 

Plant growth regulators (21) 

• Indole-3-acetic Acid 

Herbicides (3) 

• Corn gluten meal 

Repellents (29) 

• Capsaicin (red pepper) 

Floral attractants and Plant Volatiles (14) 

• 1-Octen-3-ol attractant in electric bug traps 

Insect & nematode control (18) 

• Soybean oil 

Plant pathogen & microbial control (21) 

• Sodium bicarbonate

Biochemical Pesticide Data Requirements 

(original) 


– Product Identity*** 





– Analytical methods 

– Physical & Chemical Properties 

– Submittal of samples 

31


(proposed) 

• Product Chemistry (no new studies added) 

– Product Identity and composition 

– Description of starting materials, production and 

formulation process 

– Discussion of formation of impurities 

– Preliminary analysis 

– Certified limits 

– Enforcement analytical methods 

– Physical & Chemical Properties details listed (18) 

– Submittal of samples appears in residue table 

32


(original) 

• Toxicology Tier 1 Studies, acute 

– Acute oral toxicity 

– Acute dermal toxicity 

– Acute inhalation 

– Immune response 

– Acute studies on MP & EP 

• Primary eye irritation 

• Primary dermal irritation 

– Genotoxicity studies 

33

34 


(proposed) 

• Human Health Tier 1 Studies, acute 

– Acute oral toxicity -rat 

– Acute dermal toxicity 

– Acute inhalation toxicity - rat 

– Immune response moved to Tier II 

– Acute studies on MP TGAI & EP 

• Primary eye irritation 

• Primary dermal irritation 

– Mutagenicity Testing 

• Bacterial reverse mutation test 

• In vitro mammalian cell gene mutation test


(original) 

• Toxicology Tier 1 Studies (continued) 

– Hypersensitivity study 

– Hypersensitivity incidents (reporting) 

– 90 day feeding 

– 90 day dermal 

– 90 day inhalation 

– Teratogenicity 

35


(proposed) 

• Human Health Tier 1 Studies (continued) 

– Hypersensitivity study Dermal sensitization 

– Hypersensitivity incidents (reporting) 

– 90 day oral (one species) 

– 90 day dermal -rat 

– 90 day inhalation -rat 

– Prenatal developmental - rat 

36


(original) 

• Toxicology Tier II Studies (if effects seen in Tier I) 

– Mammalian mutagenicity tests 

– Immune Response 

– Residue Studies (detailed list - actually in another table) 

• Tier III Studies (to address effects seen in Tier II) 

– Chronic exposure 

– Oncogenicity 

37

38 


(proposed) 

• Toxicology Tier II Studies (if effects seen in Tier I) 

– Mutagenicity tests – listed in vitro cytogenic tests 

– Immunotoxicity 

– Residue Studies (detailed list - actually in another table) 

– Prenatal developmental (on a second species) 

– Applicator/User Exposure studies (5 + use information) 

• Tier III Studies (to address effects seen in Tier II) 

– Chronic oral – rodent & nonrodent 

– Carcinogenicity - 2 species 

– Immune response (moved from Tier II) 

– Reproduction & fertility effects 

– Mammalian spermatogonial chromosome aberration 

– Companion animal safety


Nontarget organism, fate & expression (original) 

• Tier I - effects 

– Avian acute oral 

– Avian dietary 

– Freshwater fish LC50 

– Freshwater invertebrate LC50 


– Nontarget plant studies 

39


Nontarget organisms, & environmental fate (proposed) 

• Tier I - effects 

– Avian acute oral toxicity (option of passerine species) 

– Avian dietary toxicity (option of passerine species) 

– Freshwater fish acute toxicity 

– Freshwater invertebrate acute toxicity 

– Nontarget insect studies 


• Terrestrial plant toxicity, seedling emergence 

• Terrestrial plant toxicity, vegetative vigor 

40



41 

• Tier II – exposure (if indicated by Tier I studies) 

– U.V. absorption 

– Volatility 

– Dispenser-water leaching 

– Adsorption-desorption 

– Octanol/water partition 

– Hydrolysis 

– Aerobic soil metabolism 

– Aerobic aquatic metabolism 

– Soil photolysis 

– Aquatic photolysis 

–space 

–space

42 



• Tier II – 

– U.V. absorption: moved to product chemistry table 

– Laboratory volatilization from soil 

– Dispenser-water leaching 

– Sediment & soil Adsorption-desorption parent & degradates 

– Octanol/water partition (moved to product chemistry) 

– Hydrolysis 

– Aerobic soil metabolism (added anaerobic metabolism) 

– Aerobic aquatic metabolism (added anaerobic) 

– Photodegradation on soil 

– Photodegradation in water 

– Seedling emergency, Tier II 

– Vegetative vigor, Tier II



• Tier III – risk (if toxic and excessive exposure is likely) 

– Terrestrial wildlife testing 

– Aquatic animal testing 



43



• Tier III – risk (if toxic and excessive exposure is likely) 

– Terrestrial wildlife testing 

• Avian Reproduction 

• Wild mammal acute toxicity 

• Terrestrial field testing 

– Aquatic animal testing (listed specific tests) 

• Freshwater fish/invertebrate testing 

• Marine/Estuarine fish/invertebrate animal testing 

• Aquatic field fish/invertebrate testing 

– Nontarget plant studies (listed the 2 actual guidelines) 

– Nontarget insect testing (listed the actual guideline) 

• Field testing for Pollinators 

44

Summary: Biochemical Pesticides: 

45 

New data requirements (added to Tiers II & III to better evaluate 

unexpected positive results seen at lower tiers ): 

– Applicator/user exposure data 

– Prenatal development, 2 nd species 

– Companion animal safety data 

– Reproduction & fertility effects 

– Anaerobic metabolism studies 

– Seedling emergence and Vegetative vigor 

Revised data requirements 

– Test notes better describe when studies are needed 

– Revised tiering for immunotoxicity 

– List physical/chemical properties 

– Replaced Hypersensitivity with Dermal sensitization 

Deleted data requirements: 

– Pheromones exempted: 

• All Arthropod exempted from Non-target & Fate data 

• Straight chain Lepidopteran exempted from Human Health data

Summary 

46 

• The proposed rule revisions reflect our 

actual practices that have evolved 

through 20 years of experience 

• It provides more help for applicants and 

reviewers 

– Clearer definitions for both biochemical and 


– Better footnotes – decreases need for data waivers 

– Data requirement updates - adds new, codifies and 

revises existing, and deletes some data 

requirements 

– Describes procedures for helping applicants

Microbial & Biochemical - REBECA

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