Document ID: EPA-HQ-OPP-2015-0484-0003
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2016-07-01T04:00Z

*** FIFRA CONFIDENTIAL BUSINESS INFORMATION was removed to a separate CBI appendix ***
                            DATA EVALUATION RECORD 
Primary review by: Joel V. Gagliardi, Ph.D.
Secondary review by: John L. Kough, Ph.D.
Study Types
Product Identity (885.1100); Manufacturing Process (885.1200); Culture Collection Deposition (885.1250); Discussion of Formation of Unintentional Ingredients (885.1300); Analysis of Samples (885.1400); Certification of Limits (885.1500); Physical and Chemical Characteristics (830.6302 - 830.7300).
MRID Nos.
496088-01; 496088-02; 496088-03; 496088-04; 496088-05; 496088-06; 496088-07; xxxxxx-xx; xxxxxx-xx.
Test Material
RotStop[(R)] C Biological Fungicide containing 3.0x10[6] CFU/g Phlebiopsis gigantea strain VRA 1992.
Study Nos.
None; none; 1841/005; 1841/006; REP-247-2; REP-150-1; none.
Sponsor
Verdera Oy; Kurjenkellontie 5 B; FI-02271 Espoo; Finland.
Testing Facilities
Safepharm Laboratories Limited Shardlow Business Park Shardlow, Derbyshire England, DE72 2GD; Verdera Oy, Kurjenkellontie 5 B FI-02271 Espoo, Finland.
Titles of Reports
Biological Properties of Phlebiopsis gigantea strain VRA 1992; Manufacturing Process, Theoretical Discussion of Impurities, Analysis of Samples, and Certification of Limits; Rotstop: Determination of Accelerated Storage Stability; Rotstop: Determination of Long-Term Storage Stability; Storage Stability of Rotstop C; Tap Density of Rotstop C; Physical/Chemical Properties - Data Waivers. 
Authors
Marina Brandtberg; Olav Messerschmidt; A.J. Woolley; D.M. Mulley; P. Seiskari.
Studies Completed
April 15, 2015; April 29, 2015; January 9, 2004; December 15, 2004; August 5, 2013; September 8, 2005; April 28, 2015.
Study Summaries
RotStop[(R)] C Biofungicide is an end use product to prevent infection of newly cut conifer stumps with tree pathogens causing annosus root rots, including Heterobasidion irregulare. The unregistered active ingredient is 10% w/w Phlebiopsis gigantea strain VRA 1992, with the end-use product containing a minimum of 3x10[6] CFU/g. The CSF and product label are in agreement concerning the content of active ingredient in the product. The inert ingredients are minimal risk and have established food tolerance exemptions. The registrant has not submitted a petition for the exemption from the requirement of a tolerance for the active ingredient and given that only logged conifer stumps are treated, this is appropriate. A discussion of the manufacturing process was provided though it was not specific for products containing Phlebiopsis gigantea strain VRA 1992. An acceptable discussion of formation of unintentional ingredients was provided. Acceptable results for the analysis of samples were provided. Each batch is tested for presence of any Escherichia coli, Staphylococcus aureus or Salmonella spp. and the batch is rejected if found. The high quality of inerts also serves as a quality determinant and no toxicity or irritation is expected from the inert ingredients added. The certified limits for the active and inert ingredients are not within their respective OCSPP-recommended ranges. An acceptable description of the physical and chemical characteristics was provided but storage stability was only demonstrated through 5 months at 4-25[o]C while a claim was made of stability for 1 year. Microbial pesticides should generally include respiratory PPE and for label uses here including sprays this should be added and is currently missing.
Classifications
SUPPLEMENTAL but upgradeable - when 1) the complete manufacturing process is submitted; 2) the CSF is updated for certified limits and the missing inert; 3) inhalation PPE is added to the label; and 4) suitable storage stability data is submitted or an expiration date within 5 months of production is added to the label.
Good Laboratory Practice
These studies were not conducted according to 40 CFR part 160 and therefore are not GLP.

I. PRODUCT IDENTITY:
Product Name: Phlebiopsis gigantea strain VRA 1992.	
Trade Names: RotStop[(R)] C Biofungicide.
Name and Address of Applicant: Verdera Oy; Kurjenkellontie 5 B (P.O. Box 1); FI-02271 Espoo; Finland.	
Name and Address of Manufacturing Plant: Salutaguse Pärmitehas A.S.; Kohila Parish; Rapla 79745 Estonia; EPA Est. No. 91225-EST-001.
Common Name: Phlebiopsis gigantea.
Deposition in a culture collection: see confidential appendix.
Regulatory Status: Registered with PMRA, Canada on July 15, 2014; 14 European isolates are registered in the EU though in the UK any local strain of Phlebiopsis gigantea may be produced as a pesticide. Previously a U.S. Phlebiopsis gigantea isolate was used in woodlots and forests prior to the need for registration, at which time that use ceased.
 INTENTIONALLY ADDED INERT INGREDIENTS: see confidential appendix.
B. CHARACTERIZATION OF THE MPCA:
i) Taxonomic designation: 	Kingdom: Fungi
						Phylum: Basidiomycota
						Class: Agaricomycetes
						Order: Polyporales
						Family: Phanerochaetaceae
						Genus: Phlebiopsis
						Species: gigantea (Fr.) Julich
						Strain: strain VRA 1992
ii) Superseded names associated with the microorganism: Phlebia gigantea (Fr) Donk; Peniophora gigantea (Fr.) Massee; Phanerochate gigantea (Fr.:Fr.) Rattan; Phlebiopsis gigantea strain 92.104.
iii) Strain origin: found as a sporocarp on a stump of Pinus resinosa in Harrington, Quebec, Canada.
iv) Natural occurrence of the microorganism: Phlebiopsis gigantea like other plant decay fungi are common in boreal and temperate forested areas worldwide. Phlebiopsis gigantea grows mainly on conifer logs and stumps and causes white rot. 
v) Mode of Action: Direct interaction of Phlebiopsis gigantea hyphae disrupts hyphae of the tree pathogens Heterobasidion annosum sensu stricto, Heterobasidion parviporum Niemela & Korhonen, Heterobasidion abietinum Niemela & Korhonen, Heterobasidion irregulare (Underw.) Garbel & Otrosina, and Heterobasidion occidentale Otrosina & Garbel; superseded names of this pathogen group are Heterobasidion annosum and Fomes annosus. Heterobasidion irregulare and Heterobasidion occidentale cause annosus root rot in North America, while the others are the main cause in Europe and elsewhere. 
vi) Pest host range: the label lists the target pest as species of the Heterobasidion annosum complex including H. irregular on conifer stumps. Some other rot fungi may also be inhibited, again by direct competition.
vii) Life cycle: Fungal hyphae grow within stumps or logs and during certain parts of the year will form fruiting bodies, sporulate, then spread by wind, rain or arthropods via asexual (oidia) or sexual spores to surrounding areas. All strains of Phlebiopsis gigantea are thought to be interfertile. Genera in the fungal family Phanerochaetaceae are not reported to cause deleterious human or animal health effects. The USDA APHIS list of widely prevalent fungi has two Phanerochaetaceae, Erythricium salmonicolor (the cause of pink disease in tropical and subtropical areas  -  notably in Mississippi) and Irpex lacteus (milk-white toothed polypore) a macrofungi common to Eastern and Central North America though not a reported plant pathogen in the U.S.
viii) Growth temperature range: Very slow growth is possible at >4oC, sporulation may occur at 0-22oC while no growth occurs at 35oC. Optimum growth temperature is 28oC however Phlebiopsis gigantea may survive both lower and higher temperatures than optimum and re-grow when optimum temperatures resume.
ix) History of use: Phlebiopsis gigantea strain VRA 1992 was registered by PMRA, Canada on 7/15/2014 for identical uses.
II. MANUFACTURING PROCESS: See confidential appendix.
III. DISCUSSION OF FORMATION OF UNINTENTIONAL INGREDIENTS: see confidential appendix.
 IMPURITIES ASSOCIATED WITH TGAI: No toxic impurities at >0.1% are associated with the TGAI. Common metabolites are: 2',3',5',-trimethoxy-p-terphenyl; lup-19(22)-ene; lupa 15, 19(22)-diene; and l-asparagine. These metabolites are not thought to participate in the mode of action excluding annosus root rot pathogens from cut stumps. A literature search did not turn up any toxicity concerns for these metabolites.
IV. ANALYSIS OF SAMPLES: For seven batches of RotStop C.
TABLE 1. Analysis of samples for RotStop C containing Phlebiopsis gigantea strain VRA 1992.
                                Test Substance
                                     Limit
                                   Batch no.
                                       
                                       
                                   302RF002
                                   302RF003
                                   302RF004
                                   302RF005
                                   302RF006
                                   404RP5105
                                  404RF03205
                             Phlebiopsis gigantea
                             > 3 x 10[6] CFU/g
                                  2.6 x 10[7]
                                  2.3 x 10[7]
                                  1.0 x 10[8]
                                  6.4 x 10[7]
                                  2.6 x 10[7]
                                  1.4 x 10[7]
                                  5.4 x 10[7]
                               Residual Moisture
                                     3-7 %
                                     3.68
                                     3.58
                                     3.83
                                     3.86
                                     3.88
                                     7.82
                                     4.65
                               Ignition Residue
                                    83-89 %
                                     85.71
                                     86.54
                                     86.44
                                     85.99
                                     85.76
                                     N.T.
                                     N.T.
                                   Cell Mass
                                       %
                                     10.6
                                      9.9
                                      9.7
                                     10.2
                                     10.4
                                     N.T.
                                     N.T.
                             Escherichia coli [a]
                                 Absent in 1 g
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.T.
                                     N.T.
                           Staphylococcus aureus [b]
                                 Absent in 1 g
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.T.
                                     N.T.
                              Salmonella spp. [c]
                                Absent in 25 g
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.D.
                                     N.T.
                                     N.T.
Data from pp. 56-59 of 112, MRID xxxxxxxx-xx; N.D. = None detected at set limit; N.T. = Not tested; N/A = Not applicable. [a] ISO/TS 16649-3; [b] EVS-EN ISO 6888-3; [c] EVS-EN ISO 6579.
V.	CERTIFICATION OF LIMITS: See confidential appendix.

VI. SUMMARY OF PHYSICAL AND CHEMICAL PROPERTIES: presented in Table 3.
TABLE 3. Description of Chemical and Physical Properties for RotStop C containing Phlebiopsis gigantea strain VRA 1992.
                                   Guideline
                                   Property
                                    Result
                               Method/Reference
                                   830.6302
                                     Color
                               Cream to opaque.
                              Visual inspection.
                                   830.6303
                                Physical State
                                    Powder.
                              Visual inspection.
                                   830.6304
                                     Odor
                            Weak fungus like odor.
                             Olfactory inspection.
                                   830.6313
                                   Stability
      Not applicable, the product will not be stored in metal packaging.
                                       
                                   830.6317
                               Storage Stability
                  Stable for 5 months when stored at 4-25°C.
      There is a claim of stability for 12 months at 4+2°C but no data.
                         Most probable number on PDA.
                                   830.6319
                                  Miscibility
          Not applicable, the product is not an emulsifiable liquid.
                                       
                                   830.6320
                           Corrosion characteristics
           Not corrosive to commercial packaging when stored @ 8°C.
                              Visual observation
                                   830.7000
                                      pH
                                     6.30
                             1% solution in water.
                                   830.7100
                                   Viscosity
                 Not applicable, the product is not a liquid.
                                       
                                   830.7300
                           Density/relative density
                                  0.22 g/mL.
                                  CIPAC MT 33
Data from MRIDs 496088-03; 496088-04; 496088-05; 496088-06, 496088-07.
DEFICIENCIES: Microbial pesticides should generally include respiratory PPE and for label uses here including sprays this should be added and is currently missing; storage stability was only demonstrated through 5 months at 4-25[o]C though a claim was made of stability for 1 year.
      
                            DATA EVALUATION RECORD
Review by: Joel V. Gagliardi, Ph.D.
Secondary Review by: John L. Kough, Ph.D.
Waiver Requests
Acute Oral Toxicity and Pathogenicity (885.3050); Acute Pulmonary Toxicity and Pathogenicity (885.3150); Acute Injection Toxicity and Pathogenicity (885.3200); Acute Oral Toxicity (870.1100); Acute Dermal Toxicity (870.1200); Acute Inhalation Toxicity (870.1300); Primary Eye Irritation (870.2400); Primary Dermal Irritation (870.2500).
MRID No.
499251-01.
Test Material
RotStop[(R)] C Biological Fungicide containing 3.0x10[6] CFU/g Phlebiopsis gigantea strain VRA 1992.
Study No.
None given.
Sponsor
Kemira Agro Oy; P.O. Box 330; Helsinki, Finland.
Testing Facility
Kemira Agro Oy; P.O. Box 330; Helsinki, Finland.
Titles of Report
Tier 1: Microbial Pesticide Mammalian Toxicology Data Requirements; Satisfaction of Data Requirements with Literature/Rationale and Data Waivers.
Author
Olav Messerschmidt.
Study Completed
June 2, 2016.
Study Summary
Phlebiopsis gigantea strain VRA 1992 was originally isolated in North America and is registered for identical uses on cut conifer stumps by the PMRA. Previously a U.S. Phlebiopsis gigantea isolate was used in woodlots and forests prior to the need for registration, at which time that use ceased. In the European Union there are 14 registered isolates of P. gigantea including use of any local isolate under the registration for the UK. Literature analyzing European and North American populations indicates genetic markers are specific to either Europe or North America but a third are identical. The genetic differences are assumed to reflect adaptation to local host conifer species and climate conditions, though European and North America isolates are very similar in terms of their biology and ecology. North American (U.S. and Canadian) strains of Phlebiopsis gigantea are interfertile at approximately the same rate as when testing geographically isolated European strains and were not distinguishable morphologically. On both continents P. gigantea occupy the same and narrow ecological niche in coniferous forests. P. gigantea is a white-rot fungus decaying moribund coniferous wood and already has wide natural dispersal in coniferous forests of Europe and North America. Control of Heterobasidion irregulare and other annosus root rot spp. by stump treatment with P. gigantea is achieved by competition for space and nutrients, and does not involve production of antibiotics or other secondary metabolites. Phlebiopsis gigantea is a mesophile and does not grow near mammalian body temperature so infections are unlikely to occur. Since the 1990's P. gigantea was used commercially in Europe on several hundred thousand hectares annually. There are no known reports of any adverse effects for pesticide applicators or personnel handling P. gigantea-based products. A literature search for toxicity, metabolites and pathogenicity of P. gigantea and related organisms found no report of any adverse human health or environmental effects. Analysis of animal studies performed on Phlebiopsis gigantea strain VRA 1835, a Finnish isolate in European RotStop products, to support all EU registrations, demonstrated the low hazard potential for Phlebiopsis gigantea strains in general. However, notable toxicity on inhalation and lack of label respiratory PPE, a requirement to forestall hypersensitivity to microbial pesticides in general, means label changes are necessary. The high quality of inerts serves as a quality determinant and no toxicity or irritation is expected from the inert ingredients added.
Classification
ACCEPTABLE  -  the final label MUST SPECIFY USE OF RESPIRATORY PPE.
Good Laboratory
Practice
This was not a study so the volume was not compliant with 40 CFR Part 160.

The registrant recently participated on a European Union re-registration panel concerning use of 14 European isolates in the EU. One registration in the UK allows use of any local strain of Phlebiopsis gigantea when produced as a pesticide. Phlebiopsis gigantea strain VRA 1992 was originally isolated in North America and is registered for identical uses, on cut conifer stumps, by the PMRA. Previously a U.S. Phlebiopsis gigantea isolate was used in woodlots and forests prior to the need for registration in the 1970's, at which time that use ceased. Literature showing analysis of genetic markers between European and North American populations indicates RAMS markers are specific to either Europe or North America but 29% of them are common to both, and some Canadian isolates have the same ITS-genotype AC: an identical ITS-A allele, and the ITS-C allele which contains some differences (Vainia and Hantula 2000). These genetic differences are assumed to reflect adaptation to local host conifer species and climate conditions, i.e. differential expression of genes involved in colonization and degradation of the available woody substrate, rather than any fundamental difference between isolates from the two continents, since populations of P. gigantea in Europe and North America are very similar in terms of their biology and ecology. North American (U.S. and Canadian) strains of Phlebiopsis gigantea were shown to be interfertile at approximately the same rate as when testing geographically isolated European strains and were not distinguishable morphologically (Grillo et al. 2005). On both continents P. gigantea occupy the same and narrow ecological niche in coniferous forests. P. gigantea is a white-rot fungus specialized in decaying moribund coniferous wood and already has wide natural dispersal in coniferous forest ecosystems in Europe and North America. Control of Heterobasidion spp. by stump treatment with P. gigantea is achieved by competition for space and nutrients, and does not involve production of antibiotics or other secondary metabolites. Common metabolites are: 2',3',5',-trimethoxy-p-terphenyl; lup-19(22)-ene; lupa 15, 19(22)-diene; and l-asparagine. These metabolites are not thought to participate in the mode of action excluding annosus root rot pathogens from cut stumps. Phlebiopsis gigantea is a mesophile and does not grow above 35[o]C so infections are not likely to occur since body temperature in mammals are typically 37[o]C and higher. Since the 1990's P. gigantea was used commercially in Europe on several hundred thousand hectares annually. There are no known reports of any adverse effects for pesticide applicators or personnel handling P. gigantea-based products. A literature search of toxicity, metabolites and pathogenicity of P. gigantea and related organisms found no report of any adverse human health or environmental effects. 

Table 1. Summary of animal testing for Phlebiopsis gigantea strain VRA 1835, and isolate from Finland.
Guideline study 
Test material
Result
Equivalence
Acute Oral Toxicity and Pathogenicity  -  Rat

MRID 496088-09
4.26x10[7] CFU/Kg Phlebiopsis gigantea strain VRA 1835 in RotStop.
No mortalities or evidence of toxicity, infectivity or pathogenicity. P. gigantea was not recovered from any tissue through day 22. Rats had soft and discolored feces through 24 hours though weight gain was appropriate throughout the study. There were no organ or tissue abnormalities upon necropsy.
Not toxic, pathogenic or infective.
Acute Pulmonary Toxicity and Pathogenicity  -  Rat

MRID 496088-10
1.12x10[6] CFU/Kg Phlebiopsis gigantea strain VRA 1835 in RotStop.
No evidence of pathogenicity or infectivity. There were three male and two female mortalities in live test substance treatment groups, and a female mortality in the autoclaved test substance group at 1-48 hours post-dosing. Many treated animals had respiratory distress and other clinical signs following dosing. P. gigantea was recovered from lungs in treated animals at 1 hour but cleared by 24 hours. Weight gain was appropriate and there were no organ or tissue abnormalities upon necropsy.
Not pathogenic or infective. 

Notable pulmonary toxicity with mortalities.
Acute Intraperitoneal Injection Toxicity and Pathogenicity  -  Rat

MRID 496088-11
0.931-1.27x10[5] CFU/Kg Phlebiopsis gigantea strain VRA 1835 in RotStop.
No mortalities or evidence of infectivity or pathogenicity. Several treated animals showed transient clinical signs up to 2 hours post-dosing though none had a pyrogenic response. Treated animals lost weight on days 1-4 though by day 22 there was satisfactory weight gain. P. gigantea was not recovered from any tissue through day 22. Treated animals had white nodules on organs at necropsy likely due to the clearance process.
Not pathogenic or infective.

Transient toxicity noted.
Acute Dermal Toxicity and Pathology  -  Rat

MRID 496088-12
2000 mg/Kg of Rotstop: 3x10[7] CFU/g Phlebiopsis gigantea strain VRA 1835.
Very slight dermal irritation occurred in 4/5 females with resolution by 10 days. Treated animals were lethargic through 48 hours. Two females lost weight on day 8 and had lower weight gains by day 15. No abnormalities were noted on necropsy. LD > 2000 mg/Kg.
EPA Toxicity Category III due to the 2000 mg/Kg limit dose.
Primary Eye Irritation  -  Rabbit

MRID 496088-13
40 mg in 0.1 mL of Rotstop: 4.20x10[5] CFU Phlebiopsis gigantea strain VRA 1835 per eye.
Redness was noted in all animals with clearance after day 4 and 7 for un-rinsed eyes. Mild corneal irritation was noted in two un-rinsed eyes with clearance after 24-48 hours. The maximum average score was 10.3/110 at 24 hours declining to 5.7 at 48 hours, then 2.0 at 72-96 hours. The final animal had a score of 1 for redness until after day 7.
EPA Toxicity Category III.
Primary Dermal Irritation  -  Rabbit

MRID 496088-14
0.5 g in 0.5 mL of Rotstop: 1.07x10[7] CFU Phlebiopsis gigantea strain VRA 1835 per rabbit.
After 24 hour exposure of undamaged fur-free skin to 0.5 g RotStop there were no symptoms of erythema or edema. No animals exhibited any clinical signs of toxicity through the 72 hour observation period. Not dermally irritating.
EPA Toxicity Category IV.
Skin sensitization  -  Guinea Pig

MRID 496088-15
1:3 saline dilution of RotStop: 3x10[7] CFU/g Phlebiopsis gigantea strain VRA 1835.
There were no clinical signs or dermal irritation noted during the study. All animals gained weight. There was no sign of skin sensitization during induction and then challenge.
Not a skin sensitizer.

REFERENCES:
Grillo, R., J. Hantula and K. Korhonen. 2005. Interfertility between North American and European strains of Phlebiopsis gigantea. Forest Pathology 35:pp. 173-182.
Vainio, E.J. and J. Hantula. 2000. Genetic differentiation between European and North American populations of Phlebiopsis gigantea. Mycologia 92(3):pp. 436-446.

                            DATA EVALUATION RECORD
Review by: Joel V. Gagliardi, Ph.D.
Secondary Review by: John L. Kough, Ph.D.
Waiver Requests
Avian Oral Toxicity and Pathogenicity (885.4050); Freshwater Fish Toxicity and Pathogenicity (885.4200); Freshwater Invertebrate Toxicity and Pathogenicity (885.4240); Nontarget Plant Testing (885.4300); Nontarget Insect Testing (885.4340); Honey Bee Testing (885.4380).
MRID No.
496088-19.
Test Material
RotStop[(R)] C Biological Fungicide containing 3.0x10[6] CFU/g Phlebiopsis gigantea strain VRA 1992.
Study No.
None given.
Sponsor
Kemira Agro Oy; P.O. Box 330; Helsinki, Finland.
Testing Facility
Kemira Agro Oy; P.O. Box 330; Helsinki, Finland.
Titles of Report
Microbial Pesticide Non-Target Organisms and Environmental Fate Data Satisfaction of Data Requirements with Literature/Rationale.
Author
Olav Messerschmidt.
Study Completed
June 2, 2016.
Study Summary
Phlebiopsis gigantea strain VRA 1992 was originally isolated in North America and is registered for identical uses on cut conifer stumps by the PMRA. Previously a U.S. Phlebiopsis gigantea isolate was used in woodlots and forests prior to the need for registration, at which time that use ceased. In the European Union there are 14 registered isolates of P. gigantea used since the 1990's, including use of any local isolate under the U.K. registration. Literature analyzing European and North American populations indicates genetic markers are specific to either Europe or North America but a third are identical. The genetic differences are assumed to reflect adaptation to local host conifer species and climate conditions, though European and North American isolates are very similar in terms of their biology and ecology. North American (U.S. and Canadian) strains of Phlebiopsis gigantea are interfertile at approximately the same rate as when testing geographically isolated European strains and were not distinguishable morphologically. On both continents P. gigantea occupy the same and narrow ecological niche in coniferous forests. P. gigantea is a white-rot fungus decaying moribund coniferous wood and already has wide natural dispersal in coniferous forests of Europe and North America. Control of Heterobasidion irregulare and other annosus root rot spp. by stump treatment with P. gigantea is achieved by competition for space and nutrients, and does not involve production of antibiotics or other secondary metabolites. Phlebiopsis gigantea is a mesophile and does not grow near mammalian, avian or insect body temperatures so infections are unlikely to occur. A literature search for toxicity, metabolites and pathogenicity of P. gigantea and related organisms found no report of any adverse human health or environmental effects. Analysis of animal studies performed on Phlebiopsis gigantea strain VRA 1835, a Finnish isolate in European RotStop products, to support all EU registrations, demonstrated the low hazard potential for Phlebiopsis gigantea strains in general. Direct exposures to nontarget organisms from sprays to a freshly cut conifer stump in a logged area are expected to be minimal, especially for aquatic species. While the wood begins slowly rotting, insects associated with rotting wood would interact with fungal structures. Studies have shown certain Coleoptera interact significantly, living in chambers in the infected wood and use Phlebiopsis gigantea as a food source. Insectivores would then be able to consume insects associated with rotting conifers, be exposed to fungal structures such as spores and mycelia while foraging, and have exposure to whatever the insect consumed. There is no evidence in the literature that such exposures to animals and birds are detrimental and they regularly occur naturally. Phlebiopsis gigantea infected stumps also show reduction in egg-laying by pine weevils, leading to less potential feeding on healthy conifers and lower incidence of plant pathogens spread by them. The use of Phlebiopsis gigantea is meant to preclude more invasive and plant pathogenic fungi such as Heterobasidion irregulare. Phlebiopsis gigantea is a weak colonizer and even when delivery is optimized a minimum inoculum is needed for efficacy. Alternative treatments such as urea and borate solutions have shown significant detrimental effects to nontarget vegetation, while Phlebiopsis gigantea applications showed no apparent nontarget effects in treated areas. Testing Honey Bees exposed both through an oral solution and by contact demonstrated that a similar strain, Phlebiopsis gigantea strain VRA 1835, was not infective or pathogenic at the highest does, and was practically nontoxic by either route of administration.
Classification
ACCEPTABLE  -  no adverse effects to nontarget organisms or the environment are expected  -  EPA makes a "No effect" determination for effects to Threatened and Endangered Species.
Good Laboratory
Practice
This was not a study so the volume was not compliant with 40 CFR Part 160.

The registrant recently participated on a European Union re-registration panel concerning use of 14 European isolates in the EU. One registration in the UK allows use of any local strain of Phlebiopsis gigantea when produced as a pesticide. Phlebiopsis gigantea strain VRA 1992 was originally isolated in North America and is registered for identical uses, on cut conifer stumps, by the PMRA. Previously a U.S. Phlebiopsis gigantea isolate was used in woodlots and forests prior to the need for registration in the 1970's, at which time that use ceased. Literature showing analysis of genetic markers between European and North American populations indicates RAMS markers are specific to either Europe or North America but 29% of them are common to both, and some Canadian isolates have the same ITS-genotype AC: an identical ITS-A allele, and the ITS-C allele which contains some differences (Vainia and Hantula 2000). These genetic differences are assumed to reflect adaptation to local host conifer species and climate conditions, i.e. differential expression of genes involved in colonization and degradation of the available woody substrate, rather than any fundamental difference between isolates from the two continents, since populations of P. gigantea in Europe and North America are very similar in terms of their biology and ecology. North American (U.S. and Canadian) strains of Phlebiopsis gigantea were shown to be interfertile at approximately the same rate as when testing geographically isolated European strains and were not distinguishable morphologically (Grillo et al. 2005). On both continents P. gigantea occupy the same and narrow ecological niche in coniferous forests. P. gigantea is a white-rot fungus specialized in decaying moribund coniferous wood and already has wide natural dispersal in coniferous forest ecosystems in Europe and North America. Control of Heterobasidion spp. by stump treatment with P. gigantea is achieved by competition for space and nutrients, and does not involve production of antibiotics or other secondary metabolites. Common metabolites are: 2',3',5',-trimethoxy-p-terphenyl; lup-19(22)-ene; lupa 15, 19(22)-diene; and l-asparagine. These metabolites are not thought to participate in the mode of action excluding annosus root rot pathogens from cut stumps. Phlebiopsis gigantea is a mesophile and does not grow above 35[o]C so infections are not likely to occur since body temperature in mammals, avian and insect species are typically 37[o]C and higher. Since the 1990's P. gigantea was used commercially in Europe on several hundred thousand hectares annually. There are no known reports of any adverse effects for pesticide applicators or personnel handling P. gigantea-based products. A literature search of toxicity, metabolites and pathogenicity of P. gigantea and related organisms found no report of any adverse human health, animal health, plant health or environmental effects. 

In terms of interaction with nontarget organisms, first, the use sites should be considered. Application is by spray directly to a freshly cut conifer stump in an area being logged so there is no direct aquatic or marine exposure. Logging operations are already regulated in terms of buffer zones around water bodies to protect water quality, so there would be a distance metric as well. Birds and mammals are not expected to interact significantly with logged, treated stumps, at least not in the near term after cutting and treatment. While the wood begins rotting in the slow process initiated by infection with Phlebiopsis gigantea strain VRA 1992, insects associated with rotting wood would be attracted and interact with the growing fungal structures. It was demonstrated recently that Coleoptera such as bark beetles interact significantly, living in chambers in the infected wood and use Phlebiopsis gigantea as a food source (Hsiau and Harrington 2003). Insectivores such as other insects, small mammals and birds (Brandeis et al. 2002) would then be able to consume insects associated with rotting conifers, be exposed to fungal structures such as spores and mycelia while foraging, and have exposure to whatever the insect consumed. There is no evidence in the literature that such exposures to animals and birds are detrimental and they regularly occur naturally. A positive effect of Phlebiopsis gigantea infected stumps is a noted reduction in egg-laying by pine weevils, that may cause damage to healthy conifers directly, and spread pathogenic fungi from wood not protected by Phlebiopsis gigantea (Skrzecz 1996; Skrzecz 2001). While the above exposures are expected from pesticide use, they already occur naturally and commonly since Phlebiopsis gigantea and other wood rot fungi quickly colonize any wounded plant material, especially fresh cut tree stumps. The use of Phlebiopsis gigantea is meant to preclude more invasive fungi such as Heterobasidion irregulare, a pathogen of living plants in addition to being a rot fungus (Hunt and Cobb 1982). Phlebiopsis gigantea is a weak colonizer and even when applied to the cut stump a minimum inoculum is needed for establishment and a non-optimized delivery may allow other, including plant pathogenic, primary colonizers to take hold (Asiegbu et al. 1996). In terms of alternatives, urea and borate solutions may be used though both showed significant detrimental effects to nontarget vegetation and may have other effects, such as nutrient pollution, while Phlebiopsis gigantea applications showed no apparent nontarget effects in treated areas (Westlund and Nohrstedt. 2000). Testing Honey Bees exposed both through an oral solution and by contact demonstrated that a similar strain, Phlebiopsis gigantea strain VRA 1835, was not infective or pathogenic at the highest dose, and was practically nontoxic by either route of administration.

Table 1. Summary of animal testing for Phlebiopsis gigantea strain VRA 1835, and isolate from Finland.
Guideline study 
Test material
Result
Equivalence
Acute Oral Toxicity and Pathogenicity  -  Rat

MRID 496088-09
4.26x10[7] CFU/Kg Phlebiopsis gigantea strain VRA 1835 in RotStop.
No mortalities or evidence of toxicity, infectivity or pathogenicity. P. gigantea was not recovered from any tissue through day 22. Rats had soft and discolored feces through 24 hours though weight gain was appropriate throughout the study. There were no organ or tissue abnormalities upon necropsy.
Not toxic, pathogenic or infective.
Acute Pulmonary Toxicity and Pathogenicity  -  Rat

MRID 496088-10
1.12x10[6] CFU/Kg Phlebiopsis gigantea strain VRA 1835 in RotStop.
No evidence of pathogenicity or infectivity. There were three male and two female mortalities in live test substance treatment groups, and a female mortality in the autoclaved test substance group at 1-48 hours post-dosing. Many treated animals had respiratory distress and other clinical signs following dosing. P. gigantea was recovered from lungs in treated animals at 1 hour but cleared by 24 hours. Weight gain was appropriate and there were no organ or tissue abnormalities upon necropsy.
Not pathogenic or infective. 

Notable pulmonary toxicity with mortalities.
Acute Intraperitoneal Injection Toxicity and Pathogenicity  -  Rat

MRID 496088-11
0.931-1.27x10[5] CFU/Kg Phlebiopsis gigantea strain VRA 1835 in RotStop.
No mortalities or evidence of infectivity or pathogenicity. Several treated animals showed transient clinical signs up to 2 hours post-dosing though none had a pyrogenic response. Treated animals lost weight on days 1-4 though by day 22 there was satisfactory weight gain. P. gigantea was not recovered from any tissue through day 22. Treated animals had white nodules on organs at necropsy likely due to the clearance process.
Not pathogenic or infective.

Transient toxicity noted.
Acute Dermal Toxicity and Pathology  -  Rat

MRID 496088-12
2000 mg/Kg of Rotstop: 3x10[7] CFU/g Phlebiopsis gigantea strain VRA 1835.
Very slight dermal irritation occurred in 4/5 females with resolution by 10 days. Treated animals were lethargic through 48 hours. Two females lost weight on day 8 and had lower weight gains by day 15. No abnormalities were noted on necropsy. LD > 2000 mg/Kg.
EPA Toxicity Category III due to the 2000 mg/Kg limit dose.
Primary Eye Irritation  -  Rabbit

MRID 496088-13
40 mg in 0.1 mL of Rotstop: 4.20x10[5] CFU Phlebiopsis gigantea strain VRA 1835 per eye.
Redness was noted in all animals with clearance after day 4 and 7 for un-rinsed eyes. Mild corneal irritation was noted in two un-rinsed eyes with clearance after 24-48 hours. The maximum average score was 10.3/110 at 24 hours declining to 5.7 at 48 hours, then 2.0 at 72-96 hours. The final animal had a score of 1 for redness until after day 7.
EPA Toxicity Category III.
Primary Dermal Irritation  -  Rabbit

MRID 496088-14
0.5 g in 0.5 mL of Rotstop: 1.07x10[7] CFU Phlebiopsis gigantea strain VRA 1835 per rabbit.
After 24 hour exposure of undamaged fur-free skin to 0.5 g RotStop there were no symptoms of erythema or edema. No animals exhibited any clinical signs of toxicity through the 72 hour observation period. Not dermally irritating.
EPA Toxicity Category IV.
Skin sensitization  -  Guinea Pig

MRID 496088-15
1:3 saline dilution of RotStop: 3x10[7] CFU/g Phlebiopsis gigantea strain VRA 1835.
There were no clinical signs or dermal irritation noted during the study. All animals gained weight. There was no sign of skin sensitization during induction and then challenge.
Not a skin sensitizer.
Acute Toxicity to Honey Bees

MRID 496088-18
0.01, 0.1, 1, 10 and 100 g/bee 48-hour oral and contact study using RotStop: 1.07x10[7] CFU Phlebiopsis gigantea strain VRA 1835.
Honey Bees were observed at 4, 24 and 48 hours after exposures. No evidence of infectivity or pathogenicity were noted. Control mortality was <10% so the study data is considered valid. At the highest dose oral toxicity (16.7%) is only slightly higher than the controls (8.3%) at 48 hours. Repellency was not noted; the entire oral dose was consumed. At the highest dose contact toxicity (16.7%) is only slightly higher than the controls (10%) at 48 hours.
Not pathogenic or infective. Practically nontoxic to Honey Bees.

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