**EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY**  **NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER**  EPA Biopesticides and Pollution Prevention Division contact: Shannon Borges (703

. 305

. 7175) LidoChem, Inc. 20 Village Court Hazlet, NJ 07730 Petition Number: _______________ EPA has received a pesticide petition [Petition Number:

____________________] from LidoChem, Inc. (hereinafter “LidoChem”), 20 Village Court; Hazlet, NJ 07730 (submitted by RegWest Company, LLC: 8203 West 20th St., Suite A; Greeley, CO 80634
-4696) requesting, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180 To establish an exemption from the requirement of a tolerance for: **M icrobial pesticide**  **:** Bacillus amyloliquefaciens strain PTA
-4838 Pursuant to section 408(d)(2)(A)(i) of F FDCA, as amended, LidoChem has submitted the following summary of information, data and arguments in support of their pesticide petition. This summary was prepared by RegWest Company, LLC for LidoChem and EPA has not fully evaluated the merits of the pesticide petition. The summary may have been edited by EPA if the terminology used was unclear, the summary contained extraneous material, or the summary u nintentionally made the reader conclude that the findings reflected EPA’s position and not the position of the petitioner. **I. LidoChem**  **Petition Summary** Petition Number: _______________ 

### A. **Product Name and Proposed Use Practices** The microbial pest control product, Bacillus amyloliquefaciens strain PTA
-4838, powder formulation is a chitinase
- , protease
- and lipase
-producing soil bacterium that is efficacious as a biocontrol agent against certain pathogenic fungi , nematodes and to help establish a vigorous root system, resulting in more uniform foliage and overall enhanced plant health. **Commercial/Agricultural Uses**  Varnimo WSP may be applied alone, in combination and/or rotation with chemical fungicides as a tool for integrated disease management in bushes, crops, herbs, spices, ornamentals, shrubs, trees (forest and shade) , turf, vegetables and vines

. It offers a valuable tool for the management of chemical pesticides’ resistance through its multiple and unique modes of action. **The proposed uses are as foll ows:**  Varnimo WSP , as a fungicide and nematode suppressant, may be used on all crops, ornamentals and turf at the rate of 3
-32 ounces of product in a minimum of 25 gallons of water (drench and drip applications) and the same dilution applied per acre (foliar application). **Varnimo WSP, as a plant growth promoter, may be used in the following methods:** Transplants (including plugs): Apply Varnimo WSP to transplants by dipping or drenching, making sure the root system is thoroughly soaked. For dipping, follow the instructions for "Cutting and Root Dips" before planting transplants into soil medium. For drenching, first plant the transplants into soil medium and then follow instructions for "Drenching".2

Drenching: Apply Varnimo WSP to seedlings or to newly rooted cuttings. Drench plants with Varnimo WSP’s suspension making sure the root system is thoroughly soaked, allowing Varnimo WSP to work into the root zone. Cutting and Root Dips: Stir suspension for several minutes to ensure complete mixture and to elim inate clumps. Place rootstock in the suspension for 5
- 10 minutes, allowing time for Varnimo WSP to penetrate the root zone. For ornamentals, apply at least one follow
-up drench treatment 2
-3 weeks following initial treatment. Tree Injection: Refer to manufacturers’ instructions for individual tree injection methods/devices. New Turf Grow
- Ins: Optimal results are obtained by making the first application 2
-7 days prior to seeding, sprigging, laying sod or at the time of seeding, sprigging or sodding. Maintain soil moisture until seeds, sprigs or sod are installed. Cool Season and Warm Season Turf: Optimal results are obtained by making the first application before the first flush of roots. Hydroponics: Prepare a stock solution by adding 0.25

–0.75 ounces of Varnimo WSP, for every 50 feet of irrigation tubing, in one gallon of water. Stir product for several minutes to ensure complete suspension. Add solution to circulating water system and circulate through 3 to 5 watering cycles before clearing the system. For best results, make 2 or 3 weekly applications. Soil Incorporation: Mix Varnimo WSP into soil or soilless growing media at a rate of 0.5
-2 ounces per cubic yard. Thoroughly mix media, using mechanical mixing equipment, to ensure uniform distribution of the product. I ncorporated into soil, Varnimo WSP may be raked into growing beds prior to planting. **Residential Uses**  Varnimo WSP is a fungicide used for suppression of fungal plant diseases on home garden plants, lawn and other turfgrass areas. Application methods are limited to drench and spray (4
-8 ounces of product to 10 gallons of water with 1 gallon per every 10 square feet). Varnimo WSP is a Plant Growth Promoter used for enhancing root development and crop production on bushes, crops, herbs, spices, ornamentals, shrubs, trees (forest and shade), turf, vegetables and vines. Varnimo WSP helps establish a vigorous root system, which often results in more uniform foliage and overall enhanced plant health. Apply Varnimo WSP to field soil, to planting substrate, to solid absorbent soil amendments and directly on propagation material. **Commercial/Agricultural Seed Treatment Uses**  Varnimo ST is a biological seed treatment that provides early
-season protection against listed plant parasitic nematodes and helps to establish a vigorous root system resulting in greater yields, more uniform foliage and overall enhanced plant health. Varnimo ST is applied as a water
-based suspension of adequate volume to uniformly distribute the product over the seed surface area . The volume of suspension varies according to seed type and whether companion seed treatments are used. Application rates, depending upon the type of seed, range from 0.0 1 to 0.09 ou nce per 100 pounds of seed; 0.02 to 0.12 ounce per 140,000 soybean seeds; and 0.01 to 0.03 ounce per 1 00,000 sugar beet seeds. 

### B. **Product Identity/Chemistry 1.**  **Identity of the pesticide and corresponding residues**
**.** Bacillus amyloliquefaciens strain PTA
-4838 is a gram
-positive, nonpathogenic and harmless bacterium , and is ubiquitous in the environment; it may be isolated from the air, streams, soils and oceans and a variety of3

plants. Bacillus amyloliquefaciens strain PTA
-4838 has been described as irregular, beige, crateriform with undulate margin. Bacillus amyloliquefaciens are gram-positive rods with peritrichous flagella allowing motility. The cells often appear as long chains unlike many other Bacillus species that form as single cells. The optimal temperature for cellular growth is between 30
-40°C. Similar to other Bacillus species, Bacillus amyloliquefaciens forms endosphores allowing survival for a long period of time. Endospores appear centrally in the cells which do not have a swollen appearance. The culture is deposited in the American Type Culture Collection’s (“ATCC”) Patent Collection and identified as “New strain of Bacillus, APM
- 1 (ATCC® PTA
-4838TM). Bacillus amyloliquefaciens strain PTA
-4838 is not considered to be a human pathogen. Bacillus subtilis species, including Bacillus subtilis variety amyloliquefaciens PTA
-4838, produce proteolytic enzymes and are widely used as additives in laundry detergents. Bacillus amyloliquefaciens is used in broiler feed to improve poultry productivity by its actions at different levels. **2. Magnitude of residues**  **at the time of harvest and method used to determine the residue** 
**.** Bacillus amyloliquefaciens strain PTA
-4838 is ubiquitous in the environment, especially in soils and agricultural environments all over the world. As a result, dietary exposure to background levels of the naturally-occurring microbe may already be occurring and will likely continue to occur. Because of its ubiquitous presence in the environment, no increase in exposure to Bacillus amyloliquefaciens strain PTA
4838 resulting from the proposed pesticidal uses, when compared to existing exposure to background levels of Bacillus amyloliquefaciens strain PTA
-4838, is expected to occur. Any potentially occurring residual deposits on crops will not harm humans because the strain shows no toxicity, infectivity or pathogenicity in appropriate tests. **3. A statement of why an analytical method of detecting and measuring the levels of the**  **pesticide residue are not needed.** The acute toxicity studies discussed in Section C of this document are sufficient to show that there are no foreseeable human or domestic health hazards likely to arise from the use of the product as proposed. LidoChem is requesting an exemption from the requirement for a tolerance for any residues remaining in/on all agricultural commodities. Since enforcement of residue levels would not be needed, an enforcement analytical method is not required. 

### C. **Mammalian Toxicological Profile**  LidoChem conducted and submitted the required toxicology studies to support its petition for an exemption from the requirement of a tolerance and associated registration. Toxicology data submitted in support of the exemption from the requirement of a tolerance for Bacillus amyloliquefaciens strain PTA
-4838 included studies on the technical form and formulated product as follows: **Acute T oxicity and/or Pathogenicity**  **Varnimo ®**  **Technical OCSPP**  **Guideline 885.3050:**  The acute oral toxicity/pathology of Varnimo Technical was evaluated when administered to rats by gavage in a single high dose exposure and given an adequate post
-exposure observation period. The only observed general health finding was one male (17
-M) in Group IV

– MCPA with a slight decrease in activity on Day 0. There were no other abnormal general health findings. There were no observed abnormalities during necropsy. The test substance appeared to clear completely from brain and kidneys by Day 7, from the blood by Day 14 and from the cecum by Day 21. The lungs, spleen, liver and mesenteric lymph nodes (“ MLN ”) showed patterns of clearance through Day 21. There were no4

indications of infectivity or pathogenicity by the test substance. Varnimo Technical was determined to be non-toxic to rats when administered by oral gavage in a single dose of 3.5 x 10 8 CFU/rat. **OCSPP Guideline**  **Number 885.3150:**  The pulmonary toxicity, infectivity and pathogenicity of Varnimo Technical were evaluated when administered to rats at a single high dose exposure and an adequate post
exposure observation period. There were no abnormal general health observations. There were no observed abnormalities during necropsy. The test organism was considered completely clear from the blood and cecum by Day 7; considered completely clear from the kidneys and MLN by Day 14; considered completely cleared from the brain, lungs and spleen by Day 21; and was present in the liver on Day 14 in one out of six animals, but was clear on Day 21. Varnimo Technical was determined to be non-toxic to rats when administered by tracheal injection in a single dose of 3.1 x 10 9 CFU/g or 3.1 x 10 8 CFU/rat. **OCSPP Guideline**  **Number 885.3200:**  Varnimo Technical was evaluated to assess the acute intravenous toxicity and infectivity using a single high dose exposure and an adequate post
-exposure observation period. No abnormalities were seen during daily observations or at necropsy. There was no mortality in any group during the study. The test organism had cleared completely from the Group III (MPCA) blood by Day 3 and from the brain and the cecum by Day 7. The test organism showed a pattern of clearance from the Group III lungs, liver, spleen and M LN

. Varnimo Technical was determined to be non-toxic to rats when injected intravenously at a dose of 2.7 x 10 8 CFU/mL or 2.7 x 10 7 CFU/rat. **Varnimo ®**  **WSP OCSPP**  **Guideline Number**  **870.1100 :**  Varnimo WSP was evaluated for acute oral toxicity potential in femal e albino rats when administered as a gavage dose at 5000 mg/kg
. No mortality occurred during the study. There were no clinical signs of toxicity during the study. Animals exhibited weekly weight gain during the study. Gross necropsy conducted at termination revealed no observable abnormalities. The acute oral LD50 was determined to be greater than 5000 mg/kg. **OCSPP Guideline**  **Number 870.1200:**  Varnimo WSP was evaluated for dermal toxicity potential and relative skin irritancy when a single dose moistened with 0.5 mL of deionized (“DI”) water/g test substance, at 5050 mg/kg, was applied to the intact skin of albino rats. No mortality occurred during the study. There were no clinical signs of toxicity at any time throughout the study. Signs of dermal irritation included erythema, atonia, desquamation and alopecia on Days 7
- 14. Animals exhibited weekly weight gain, except for one that failed to gain weight between Days 0 and 7. Gross necropsy conducted at study termination revealed no observable abnormalities. The LD50 was determined to be greater than 5050 mg/kg

. **OCSPP Guideline**  **Number 870.1300:**  Varnimo WSP was evaluated for acute inhalation toxicity potential in albino rats. Pre
-study trial assays were conducted to determine if aerosolizing the test substance i nto the exposure chamber would produce an acceptable concentration of at least 2 mg/L with mass median aerodynamic diameter (“MMAD”) between 1 and 4 μm. Prior to aerosolization in some assays, the test substance was heat
-dried and/or ground. An aerosol was generated using a Venturi Aspirator connected to a 500 L exposure chamber. No attempt resulted in an appropriate exposure concentration with an appropriate correspond MMAD. Due to the nature of the test substance, an exposure concentration could not be achieved that would provide meaningful acute inhalation toxicity data. Based on the laboratory report, LidoChem submitted a waiver request for this data requirement. **OCSPP Guideline**  **Number 870.2400:**  An acute eye irritation study was conducted on three albino rabbits using Varnimo WSP. The test substance, 100 mg, was placed into the conjunctival sac of the right eye of each animal selected for testing. All treated eyes were washed with room temperature deionized (“DI”) water for one minute immediately after recording the 24
-hour observation. There were no positive effects exhibited in any eyes after treatment. The test substance is rated non
-irritating.5

**OCSPP Guideline**  **Number 870.2500:**  A primary dermal irritation study was conducted on three albino rabbits using Varnimo WSP. There was one intact test site per animal. Each test site was treated with 500 mg of Varnimo WSP moistened with 0.5 mL of deionized (“DI”) water and covered with semi
-permeable dressing. Test substance was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at 1, 24, 48 and 72 hours after removal of the dressings. Based on 0.2 PII, the test substance is rated slightly irritating. Based on scores at only the 72
-hour observation, the test substance is assigned Toxicity Category IV. The death of one animal on Day 3 of the study is considered unrelated to administration of the test substance. 

### D. **Aggregate Exposure 1.**  **Dietary exposure i.**  **Food** Bacillus amyloliquefaciens strain PTA
-4838 is ubiquitous in the environment, especially in soils and agricultural environments. As a result, dietary exposure to background levels of the naturally
-occurring microbe may already be occurring and will likely continue to occur. Because of its ubiquitous presence in the environment, no increase in exposure to Bacillus amyloliquefaciens strain PTA
-4838, resulting from the proposed pesticidal uses, is expected when compare to existing exposure to background levels of Bacillus amyloliquefaciens strain PTA
-4838. As previously discussed, dietary exposure to the naturally
-occurring microbe may already be occurring and will likely continue to occur. Notably, similar Bacillus subtilis strains are used internationally in the production of food
-grade products and in fermented foods in Japan and Thailand. Reports in the literature implicating Bacillus subtilis sp. i n food
-borne illness do not describe any pathogen or toxin production, but rather simple spoilage from Bacillus subtilis growth in dough. Such low
-quality dough would not be suitable for bread production by commercial bakeries , therefore this particular food exposure scenario is unlikely and the human risk negligible. An extensive literature search yielded no reliable reports of adverse effects on non-target terrestrial plants. One on-line report identified Bacillus amyloliquefaciens as infecting an onion in cold storage. The temperature of the storage unit was 0
- 1°C. It has been established that Bacillus amyloliquefaciens remains inactive at temperatures below 15°C or above 50°C. Eight bacterial isolates were found all to infect longitudinally
-cut onions at 30°C. LidoChem concludes this report is not reliable as the bacteria would have remained dormant during cold storage and the isolation and identification of the Baci llus amyloliquefaciens was incidental or incorrect. The risk posed to adults, infants and children from food
-related exposure to Bacillus amyloliquefaciens strain PTA
-4838 is minimal due to the demonstrated lack of acute oral toxicity/pathogenicity associated with the microbial pesticide. Based on the submitted data, there are no dietary risks exceed ing regulatory levels of concern. **ii. Drinking**  **water** Bacillus amyloliquefaciens strain PTA
-4838 is ubiquitous in the environment and exposure to the microbe through drinking water may already be occurring and will likely continue to occur. While the proposed and existing use sites do not include direct application to aquatic environments, the intended use of Bacillus subtilis treatment of growing crops or seed for the control of plant disease might result in the potential for drift or runoff that might reach surface or ground waters. Bacillus subtilis species are not recognized aquatic bacteria and are not expected to grow in aquatic environments. Municipal drinking water treatment processes and deep water wells both further reduce any such residues. Even if oral exposure to this ubiquitous microbe should occur through drinking water, due to its demonstrated lack of acute oral toxicity/pathogenicity, it is likely that no harm will result from such exposure.6
**2. Non** **- dietary exposure**  The pesticide uses of Bacillus amyloliquefaciens strain PTA
-4838 , as proposed , do not increase in a significant way the potential for non-dietary, non-occupational exposure to its residues f or the general population, including infants and children, because Bacillus amyloliquefaciens strain PTA
-4838 is naturally
occurring and ubiquitous in the environment and because it is likely populations have been previously exposed, and will likely contin ue to be exposed, to background levels of the microbe. Neither such common human exposure to Bacillus amyloliquefaciens strain PTA
-4838 naturally present in soils, waters and plants, nor exposures associated with similar Bacillus subtilis strains used internationally in producing food
-grade products and fermented foods, have resulted in reports of infections in humans (which consistently are reported only in otherwise
-compromised individuals) those reports are most notable for their rare and exceptional nature. We believe evaluation of the high
-dose Tier 1 acute toxicity and pathogenicity tests will result in the assignment of Toxicity Category IV (least toxic) and determinations of not infective and not pathogenic will be concluded for all exposure routes. No toxicological end points of concern have been identified. There are no dietary endpoints that exceed regulatory levels of concern. Any additional exposure to the microbe resulting from residues attributable to Bacillus subtilis var. amyloliquefaciens strain PTA
-4838 pesticide use , will not result in additional aggregate non-occupational risk from dermal and inhalation exposures. Based solely on non-occupation exposure scenarios , regular occupational exposures associated with this active ingredient pose negligible risk. 

### E. **Cumulative Effects** No mechanism of toxicity in mammals has been identified for Bacillus amyloliquefaciens strain PTA
-4838. Therefore, no cumulative effect with other related organisms is anticipated, because the available data demonstrate a lack of toxicity/pathogenicity potential for the active ingredient, adverse dietary effects are unlikely. 

### F. **Safety Determination 1.**  **U.S. population** Based on the acute toxicity information discussed in 3.C., there is a reasonable certainty that no harm wil l result to the United States population from aggregate exposure to residues of Bacillus amyloliquefaciens strain PTA
-4838. See “2. I nfants and Children” for rationale support

. **2. Infants and children**  Based on the acute toxicity information discussed in 3.C. , there is a reasonable certainty that no harm will result to the United States population, including infants and children, from aggregate exposure to residues of Bacillus amyloliquefaciens strain PTA
-4838. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. This is based on the available data on Bacillus subtilis species, including Bacillus amyloliquefaciens strain PTA
-4838, that demonstrate a lack of toxicity/pathogenicity potential. There are no threshold effects of concern and therefore, the additional tenfold margin of safety for infants and children is unnecessary. The need to consider consumption patterns, special susceptibility and cumulative effects is unnecessary when dealing with pesticides with no demonstrated significant adverse effects. 

### G. **Effects on the Immune and Endocrine Systems** Bacillus amyloliquefaciens strain PTA
-4838 is a ubiquitous organism in the environment that is non-toxic to mammals. To date, there is no evidence to suggest that Bacillus amyloliquefaciens strain PTA
-4838 affects the immune system, functions in a manner similar to any known hormone, or that it acts as an endocrine disruptor. The submitted toxicity/pathogenicity studies in rodents indicate that, following several routes of exposure, the immune system is intact and able to process and clear the active ingredient. Therefore, it is unlikely that this organism will have estrogenic or endocrine effects.7



### H. **Existing Tolerances** There are no existing tolerance exemptions or tolerances established for Bacillus amyloliquefaciens strain PTA
-4838. 

### I. **International Tolerances** No Codex maximum residue level (“MRL”) exists for Bacillus amyloliquefaciens strain PTA
-4838.