                  Federal Food, Drug, and Cosmetic Act (FFDCA)
              Considerations for Pseudomonas fluorescens strain D7

                                Docket ID Number: EPA-HQ-OPP-2013-0569
                                               Date: August 22, 2014

Section 408(c)(2)(A)(i) of FFDCA allows the EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if the EPA determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of FFDCA defines "safe" to mean that "there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information." This includes exposure through drinking water and in residential settings but does not include occupational exposure. Pursuant to FFDCA section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, the EPA must take into account the factors set forth in FFDCA section
408(b)(2)(C), which require the EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance exemption, and to "ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue...." Additionally, FFDCA section 408(b)(2)(D) requires that the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."

The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, the EPA determines the toxicity of pesticides. Second, the EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.

I. Summary of Petitioned-for Tolerance Exemption

In the Federal Register of September 12, 2013 (78 FR 56185) (FRL-9399-7), the EPA issued a notice pursuant to FFDCA section 408(d)(3), 21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance petition (PP 2F8103) by Northwest Agricultural Products  821 South Chestnut Avenue, Pasco, WA 99301. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of Pseudomonas fluorescens strain D7 in or on growing crops and rangeland. The notice referenced a summary of the petition prepared by the petitioner, Northwest Agricultural Products, which is available in Docket ID Number EPA-HQ-OPP-2013-0569 via http://www.regulations.gov. While rangeland is not a food commodity and "growing crops" is not a recognized commodity term used by the Agency in tolerance actions, the Agency is interpreting the petitioner's request as seeking a tolerance exemption for "all food commodities."  The term "growing crops" is quite broad and does not limit that types of food commodities that it covers; therefore, the Agency believes a reasonable interpretation of that term allows for establishment of an exemption from the requirement of a tolerance for "all food commodities."    
 
One comment concerning the general impact of pesticides on bees was filed in the Pseudomonas fluorescens D7 docket.  The comment is not relevant to this tolerance action as the comment does not concern the safety of this microbe or have any bearing on the Agency's assessment under section 408 of the FFDCA.   



II. Toxicological Profile

Consistent with section 408(b)(2)(D) of FFDCA, the EPA reviewed the available scientific data and other relevant information on Pseudomonas fluorescens strain D7, and considered its validity, completeness, and reliability, as well as the relationship of this information to human risk. The EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.

A.  Overview of Pseudomonas fluorescens strain D7 

The D7 EP is for use as an herbicide in the United States in field applications for suppression of downy brome (cheatgrass), medusahead, Japanese brome, and jointed goatgrass on fields of turf and grasses grown for seed, alfalfa; wheat, barley, triticale, oat, and rangeland. It is for application as a seed treatment and by ground or aerial spray to listed field sites.  The D7 EP is to be applied directly to the soil surface, in autumn or spring when temperatures are cooler, and the plants are in preemergence.
P. fluorescens occurs naturally in soil and water (Ref. 1), and strain D7 is a root-associated bacterium which was isolated originally from winter wheat roots from the West Coast region of the United States.

B.  Microbial Pesticide Toxicology Data Requirements

All applicable mammalian toxicology data requirements supporting the request for an exemption from the requirement of a tolerance for residues of Pseudomonas fluorescens strain D7 in or on all food commodities have been fulfilled with data submitted by the petitioner or scientific rationale. The toxicity and pathogenicity tests (dermal, toxicity) and irritation tests (acute eye and primary dermal irritation) that address potential routes of exposure to the active ingredient are all classified in Toxicity Category IV (see below or Ref. 2, 3 and 4)  and revealed little to no toxicity attributed to Pseudomonas fluorescens strain D7.  Finally, the petitioner reported that no hypersensitivity incidents occurred during the research, development, and testing of this active ingredient.

The overall conclusions from all toxicological information submitted by the petitioner are briefly described below, and more in-depth synopses of study results can be found in the associated draft Biopesticides Registration Action Document (BRAD) for Pseudomonas fluorescens strain D7, provided as a reference in Section VII of this document (Ref. 2).

   1. Acute Injection Toxicity/Pathogenicity  -  Rat (MRID 49349701) 
A study was repeated to re-evaluate the acute intravenous toxicity infectivity and pathogenicity, of a microbial pest control agent (MPCA), Pseudomonas fluorescens D7, at a single exposure of 10[6] and 10[7] doses. The MPCA test substance, inactivated MPCA, or sterile filtrate was injected into the tail vein of one of four groups of rats. A control group (untreated) was conducted concurrently. The animals were observed frequently on day of dosing for mortality and signs of pharmacological and/or toxicological effects and once 
daily thereafter for 21 days. Tissue and blood samples taken at interim sacrifices from the group receiving the active test substance were cultured to provide quantitative measurements of the test microbe's clearance pattern. There was no mortality in any group during the study. During observations for clinical signs, all animals appeared normal for the duration of the study. The gross necropsy conducted at termination of the study revealed no observable abnormalities. The test organism had cleared completely from Groups IV and V (MPCA) blood, kidneys, mesenteric lymph nodes, lungs, and brain, liver, spleen and cecum contents by Day 7. After two consecutive interim sacrifices showed no growth of test organism in the organs plated, no further tissue samples were taken. The test substance Pseudomonas fluorescens D7 was determined to be non-toxic 
and not pathogenic or infective to rats when injected intravenously at a dose of 3.4 x 107 CFU/rat or 3.7 x 106 CFU/rat.  EPA determined this study to be acceptable


This study (MRID 49349701) addresses EPA recommendations to repeat an acute injection toxicity study using a lower dose of the microorganisms that includes additional controls such as filtered culture medium, and autoclaved medium to account for the observed injection results.  This study supersedes the previously submitted acute injection toxicity/pathogenicity in rat study (MRID 48966402) that tested Pseudomonas fluorescens strain D7 and showed adverse effects to rats following a single intravenous injection administration of 1.3 x 10[8] CFU/rat including mortality, abnormal clinical signs; transient weight loss, abnormal gross necropsy findings, and altered organ weights.  

   2. Acute Oral Toxicity/Pathogenicity  -  Rat (MRID 48966403)
Sprague-Dawley rats, 8 week old, (12/sex) were given a single oral dose of MPCA Pseudomonas fluorescens strain D7 of 1.3 X 109 CFU/animal. The animals were observed three times on day 0 after dosing and daily through day 21 with interim scheduled sacrifices on Days 3, 7, and 14.  Five males and five females were treated with autoclaved Pseudomonas fluorescens strain D7 as inactive MPCA controls, two untreated animals per sex were used as "shelf controls," and two animals per sex were designated as untreated controls.

No observable abnormalities were found during observations for clinical signs or at necropsy, and there were no toxicologically relevant differences between the body weights of the treated animals and those of controls.  Relative to their respective controls, MPCA-treated females had increased relative liver weights (+16.0%; p<0.05), and MPCA-treated males had increased relative spleen weights (+16.7%; p<0.05), and these differences correlated with increased mean absolute weights of these same organs (+11.7% and +27.1% for MPCA-treated female absolute liver and MPCA-treated male absolute spleen weights, respectively).  No CFUs (or in one instance a single individual count of <100 CFU/g) were recovered from the brain, lungs, liver, kidneys, or lymph nodes of any active-treated animal.  Pseudomonas fluorescens strain D7 was found in feces, urine, and cecum contents of MCPA-treated animals and appeared to be completely cleared by day 14 following oral administration to rats.  Based on the results of this study, Pseudomonas fluorescens strain D7 does not appear to be toxic, infective, and/or pathogenic in rats, when dosed at 1.3 X 109 CFU/animal. The EPA rated this study as acceptable.


   3. Acute Pulmonary Toxicity/Pathogenicity  -  Rat (MRID 48966404)
Groups of 12 week old Sprague Dawley rats (3/sex/Group) were exposed by the intratracheal route to Pseudomonas fluorescens strain D7 in sterile PBS at a constant dose volume of 0.1 mL/ animal and a dose of 4.6 x 108 cfu/animal.  Eight males and eight females were treated with autoclaved Pseudomonas fluorescens strain D7 as autoclaved controls; five males and five females were not treated and used as untreated control; and four males and four females were not treated and used as shelf controls.  The animals were then observed for up to 21 days, with interim sacrifices on day 0 (all groups) and days 3, 7, and 14 (active-MCPA group).   

There were no test substance related clinical signs, gross necropsy findings, or differences in organ weights.  Two males in the active test material treated group sacrificed on day 3 lost weight and one female in autoclaved test material treated group sacrificed on day 21 did not gain weight during the first week but gained weight by day 14.  All other animals gained weight prior to scheduled sacrifice.  The test organisms were not seen or were present at < 100 cfu/g in lungs from the animals sacrificed after dosing on day 0.  The test organisms were not seen or were present at < 100 cfu/g in blood, brain, lungs, spleen, liver, kidneys, mesenteric lymph nodes, and cecum content removed from animals sacrificed on days 3 and 7. Based on these results, Pseudomonas fluorescens strain D7 does not appear to be toxic, infective, and/or pathogenic in rat, when dosed at 4.6 x 108 cfu /animal. The EPA rated this study as acceptable.

   4. Acute Dermal Toxicity/ Pathogenicity  -  Rabbit (MRID 48966405)
Five male and five female New Zealand White rabbits, 16-18 weeks old, were dermally exposed to a 2000 mg/kg bw dose of Pseudomonas fluorescens strain D7 moistened with 2.0 mL of deionized water/g test substance for 24 hours to an area of approximately 10% of body surface area. Following exposure, the animals were observed for a period of 14 days. All animals survived and had no abnormal systemic clinical signs during the study. Very slight to well-defined Erythema was noted on all dose sites on day 1 and resolved by day 4.  With the exception of one female that lost weight during the second week, all animals gained weight throughout the study.  No observable abnormalities were found at necropsy.  The dermal LD50 for males was greater than 2000 mg/kg bw; for females was greater than 2000 mg/kg bw; and for both sexes combined was greater than 2000 mg/kg bw.  Based on the results of this study, Pseudomonas fluorescens Strain D7 is of low toxicity.  EPA classified the acute dermal toxicity of Pseudomonas fluorescens strain D7 as Toxicity Category IV and rated this study as acceptable.

   5. Primary Eye Irritation  -  Rabbit   (MRID 49100801)
100 mg of undiluted Pseudomonas fluorescens strain D7 (Batch No. 201109210301; purity 95%; pH not reported) was instilled as supplied into the conjunctival sac of the right eye of three male and three female New Zealand White rabbits.  Untreated left eyes served as controls.  Animals were observed at 1, 24, 48, and 72 hours after test material instillation.  Irritation was scored by the method of Draize and classified by the system of Kay and Calandra.

No corneal opacity, iritis, or conjunctival irritation was noted on any rabbit throughout the study. Pseudomonas fluorescens strain D7 was not irritating to the eye and EPA classified the acute eye irritation as Toxicity Category IV for primary eye irritation. The EPA rated this study as acceptable.

   6. Primary Dermal Irritation  -  Rabbit   (MRID 49100802)
Three male and three female New Zealand White rabbits were dermally exposed to 500 mg of Pseudomonas fluorescens strain D7 moistened with 1.0 mL of DI water for 4 hours on an approximately 2.5 x 2.5 cm area of the body surface.  The animals were observed at 1, 24, 48, and 72 hours after patch removal.  Irritation was scored by the method of Draize. No dermal irritation was noted on any animal during the study.   The primary irritation index was 0.0. Pseudomonas fluorescens strain D7 was not irritating and EPA classified the dermal irritation toxicity as Toxicity Category IV. The EPA rated this study as acceptable.

6. Hypersensitivity incidents
The petitioner reported that no hypersensitivity incidents, including immediate-type or delayed-type reactions of humans and domestic animals, occurred during research, development, or testing of Pseudomonas fluorescens strain D7 (Ref. 2).

III.  Aggregate Exposure

In examining aggregate exposure, section 408 of FFDCA directs EPA to consider 
available information concerning exposures from the pesticide residue in food and all other nonoccupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

Food Exposure and Risk Characterization: The EPA found that increased dietary exposure to Pseudomonas fluorescens strain D7 a naturally occurring bacterium is anticipated to be negligible For the proposed use of Pseudomonas fluorescens strain D7 as an herbicide, the applications are made before crop plants emerge, or as seed treatment and consequently oral exposure to residues from such use is expected to be minimal.  Pseudomonas fluorescens strain D7 and other closely related Pseudomonas fluorescens bacteria already exist in the soil environment.  The EPA concluded that the risk posed to adults, infants, and children is likely to be minimal because of the low acute oral toxicity/pathogenicity potential of Pseudomonas fluorescens strain D7. 

Drinking Water Exposure and Risk Characterization: Exposure to residues of Pseudomonas fluorescens strain D7 in consumed drinking water is not likely to increase because there are no use sites for the pesticide with direct applications to water.  There is a possibility of spray drift from aerial applications or runoff of prepared fields and rangelands into surface waters. Ground water is not expected to have significant exposure to Pseudomonas fluorescens strain D7since, like other microorganisms, this microbial pesticide would likely be filtered out by the particulate nature of many soil types. If it were to be transferred to surface or ground waters that are intended for eventual human consumption (e.g., through spray drift or runoff) and directed to wastewater treatment systems or drinking water facilities, it likely would not survive the conditions water is subjected to in such systems or facilities, including chlorination, pH adjustments, filtration, and/or occasionally high temperatures (Ref 5, 6 and 7). In the remote likelihood that Pseudomonas fluorescens strain D7 is present in drinking water (e.g., water not subject to treatment systems or facilities), its target pest specificity and available data indicate no toxicity and/or pathogenicity is likely to occur with any drinking water exposure to Pseudomonas fluorescens strain D7 that results from pesticide applications made in accordance with good agricultural practices (see additional discussion in Section II of this document).

Non-occupational, Residential Risk Characterization: Given that Pseudomonas fluorescens strain D7 use sites do not include residential settings and because the bacterium is naturally-occurring  EPA determined that non-occupational exposure to the bacterium is unlikely. Repeated exposures to the Pseudomonas fluorescens strain D7 microorganism from pesticidal applications do not exceed EPA's level of concern, particularly in light of available data that demonstrate Pseudomonas fluorescens strain D7  is not toxic (acute dermal toxicity and acute pulmonary toxicity/ pathogenicity), is non-irritating (primary dermal irritation), and is not pathogenic when used as labeled in accordance with good agricultural practices (acute pulmonary toxicity/pathogenicity, and acute injection toxicity/pathogenicity). 

IV. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance exemption, the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."  The EPA has not found Pseudomonas fluorescens strain D7 to share a common mechanism of toxicity with other substances. Pseudomonas fluorescens strain D7 does not appear to be toxic to humans via dietary, dermal and pulmonary exposure. For the purposes of the tolerance action, therefore, the EPA has assumed that Pseudomonas fluorescens strain D7 does not have a common mechanism of toxicity with other substances. Thus, section 408(b)(2)(D)(v) of the FFDCA does not apply. For information regarding the EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the EPA's website at http://www.epa.gov/pesticides/cumulative.
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V.  Determination of Safety for the United States Population, Infants and Children

In considering the establishment of a tolerance or tolerance exemption for a pesticide chemical residue, FFDCA section 408 (b) (2) (C) provides that the EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity. In addition, FFDCA section 408(b) (2) (C) provides that the EPA shall apply an additional tenfold (10X) margin of exposure (safety) for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure unless the EPA determines that a different margin of exposure (safety) will be safe for infants and children. This additional margin of exposure (safety) is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the default value of 10X or uses a different additional safety factor when reliable data available to the EPA support the choice of a different factor.

Based on the acute toxicity and pathogenicity data/information discussed in section II above (and Ref. 2), the EPA concludes that there are no threshold effects of concern to infants, children, or adults when Pseudomonas fluorescens strain D7 is used as labeled in accordance with good agricultural practices. As a result, the EPA concludes that no additional margin of exposure (safety) is necessary to protect infants and children and that not adding any additional margin of exposure (safety) will be safe for infants and children.

Moreover, based on the same data/information and EPA analysis as presented directly above, the Agency is able to conclude that there is a reasonable certainty that no harm will result to the United States population, including infants and children, from aggregate exposure to the residues of Pseudomonas fluorescens strain D7 when it is used -- as labeled and in accordance with good agricultural practices -- as an herbicide. Such exposure includes all anticipated dietary exposures and all other exposures for which there is reliable information. The EPA has arrived at this conclusion because, considered collectively, the data and information available on Pseudomonas fluorescens strain D7 do not demonstrate toxic or pathogenic potential to mammals, including infants and children. 

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VI. Conclusions

The EPA concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of Pseudomonas fluorescens strain D7. Therefore, an exemption from the requirement of a tolerance is established for residues of Pseudomonas fluorescens strain D7 in or on all food commodities when applied or used in accordance with label directions and good agricultural practices.

VII. References


   1. Palleroni, N. J. 2005.  Pseudomonas. Pp. 323  -  379. In: Bergey's Manual of Systematic Bacteriology, Vol. 2. D. J. Brenner, N. R. Krieg, and J. T. Staley (Eds.) Springer, New York, New York

   2. Biopesticides Registration Action Document (BRAD) for Pseudomonas fluorescens strain D7


   3. U.S. EPA. 2014a.  Memorandum from I. Barsoum (OPP/BPPD) to S. Cerrelli, Re:  Review of Acute Toxicity/Pathogenicity study for section 3 registration of the TGAI, Fluorescens Technical, and the EP, D7, containing the active ingredient Pseudomonas fluorescens strain D7.  Dated April 15, 2014.

   4. U.S. EPA. 2014b.  Memorandum from I. Barsoum (OPP/BPPD) to S. Cerrelli, Re:  Review of Acute Toxicity/Pathogenicity study for section 3 registration of the TGAI, Fluorescens Technical, and the EP, D7, containing the active ingredient Pseudomonas fluorescens strain D7.  Dated April 22, 2014.

   5. Centers for Disease Control and Prevention. 2009. Drinking Water - Water Treatment. Available from http://www.cdc.gov/healthywater/drinking/public/water_treatment.html.

   6. U.S. EPA. 2004. Primer for Municipal Wastewater Treatment Systems. EPA 832-R-04-001. 
Available from http://www.epa.gov/npdes/pubs/primer.pdf

   7. DeFelice K, Wollenhaupt N, Buchholz D. 1993. Aquifers and Soil Filter Effect. Available from http://extension.missouri.edu/p/WQ24.








