Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Pseudomonas chlororaphis strain AFS009 

                    Docket ID Number: EPA-HQ-OPP-2016-0284
                              Date: June 1, 2017
                                       
Section 408(c)(2)(A)(i) of FFDCA allows the U.S. Environmental Protection Agency (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 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, EPA must take into account the factors set forth in FFDCA section 408(b)(2)(C), which require EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance or 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 EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."
EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, for microbial pesticides, EPA determines the pathogenicity and toxicity potential of the pesticide in tiered testing. Second, EPA examines exposure to the pesticide through food, drinking water, and other exposures that occur as a result of pesticide use in residential settings, as well as other non-occupational exposure to the substance.
I. Summary of Petitioned-for Tolerance Exemption
In the Federal Register of June 22, 2016 (81 FR 40594), EPA issued a document pursuant to FFDCA section 408(d)(3), 21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance petition (PP 5F8410) by AFS009 Plant Protection, Inc., 104 T.W. Alexander Dr., Building 18, Research Triangle Park, NC 27709. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of Pseudomonas chlororaphis subsp. aurantiaca strain AFS009 in or on all food commodities. That document referenced a summary of the petition prepared by the petitioner AFS009 Plant Protection, Inc., which is available in Docket ID Number EPA-HQ-OPP-2016-0284 via http://www.regulations.gov. One general comment opposing the establishment of the exemption was received.  
Since the time the original notice of filing was published, the petitioner provided additional data on the identity of the active ingredient to EPA. After reviewing these data, EPA now considers the correct identity of the active ingredient to be Pseudomonas chlororaphis strain AFS009 and not Pseudomonas chlororaphis subsp. aurantiaca strain AFS009. In order to give the public an opportunity to comment on this new information, EPA republished its receipt of this tolerance exemption petition filing with an updated and accurate description in the Federal Register of December 20, 2016 (81 FR 92758) and placed a revised petition from AFS009 Plant Protection, Inc. into the docket. No comments were received on this latter notice.  

II. Toxicological Profile
Consistent with FFDCA section 408(b)(2)(D), EPA reviewed the available scientific data and other relevant information on Pseudomonas chlororaphis strain AFS009 and considered its validity, completeness, and reliability, as well as the relationship of this information to human risk. EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.
The overall conclusions from all toxicological information submitted by the petitioner are briefly described below. More in-depth synopses of study results can be found in the risk assessment (Ref. 1) and other supporting science document (Ref. 2).

A. Overview of Pseudomonas chlororaphis strain AFS009

Pseudomonas chlororaphis strain AFS009 is a Gram-negative, fluorescent, Pseudomonad bacterium originally isolated from cotton plant roots in Texas. Fluorescent Pseudomonads were discovered when wheat fields resistant to root rots, including from Pythium and "Take-all root rot" (Gaeumannomyces graminis), were found to harbor these microbes on their roots while susceptible fields lacked them. Ongoing research identified certain antifungal compounds known as phenazines, namely phenazine-1-carboxylic acid (CAS No. 2538-68-3), that strongly correlated with disease control (Ref. 3). Pseudomonas chlororaphis strain AFS009, like other fluorescent Pseudomonads, colonizes the roots of plants and competes for niches plant-pathogenic fungi may also occupy, in the process producing various inhibitory metabolites that also affect these fungi. 

Environmental fluorescent Pseudomonas species, including Pseudomonas chlororaphis, occur naturally in the environment and on food, and no foodborne disease outbreaks or toxin production from Pseudomonas chlororaphis in food or feed have been reported. In fact, other strains of Pseudomonas chlororaphis have been investigated as inhibitors of foodborne pathogens.

B. Microbial Pesticide Toxicology Data Requirements

Acute toxicity (acute oral, inhalation, and dermal toxicity) and irritation tests (acute eye and
primary dermal irritation) performed with Pseudomonas chlororaphis strain AFS009 addressed potential routes of exposure to the active ingredient and reveal no toxicity or irritation attributed to Pseudomonas chlororaphis strain AFS009 (Toxicity Category IV) (Refs. 1 and 4). In the acute oral toxicity/pathogenicity study performed with Pseudomonas chlororaphis strain AFS009, there was no evidence of toxicity, pathogenicity, or infectivity when rats were administered 3.73 x 10[9] colony-forming units (CFU) per rat by oral gavage. Scientific rationale for the acute pulmonary toxicity/pathogenicity and the acute injection toxicity/pathogenicity data requirements was determined to be adequate to support waiving these two studies, based on the above findings and because the microbe grows best below 86°F (30°C) and in the presence of oxygen and is unlikely to grow or metabolize at higher temperatures or with oxygen limitations.

In light of the adequacy of the toxicological data, scientific rationale, and literature (Refs. 3, 5, and 6) provided by the petitioner, EPA did not require toxicological testing at higher tiers. Based on animal testing of Pseudomonas chlororaphis strain AFS009, no toxicity, irritation, infectivity, pathogenicity or other adverse effects attributed to Pseudomonas chlororaphis strain AFS009 are expected.

1. Acute Oral Toxicity/Pathogenicity  -  Rat (Harmonized Guideline 885.3050; Master Record Identification Number (MRID No.) 495680-02). An acceptable acute oral toxicity and pathogenicity study demonstrated that Pseudomonas chlororaphis strain AFS009 is not toxic, infective, or pathogenic by oral gavage of 3.73 x 109 CFU/rat. The test substance cleared from most tissues by day 14. While there was low detection in mesenteric lymph nodes of some animals on days 14 and 21, the 21-day study showed a distinct pattern of clearance from the gastrointestinal tract with no signs of infectivity, pathogenicity, or toxicity. (Ref. 1).

2. Acute Oral Toxicity  -  Rat (Harmonized Guideline 870.1100; MRID No. 495680-03). An acceptable acute oral toxicity study demonstrated Pseudomonas chlororaphis strain AFS009 is not toxic to female rats when dosed at 5,000 mg/Kg body weight. The oral no observed adverse effect level (NOAEL) for female rats was greater than 5,000 mg/Kg body weight (Toxicity Category IV). (Ref. 1).

3. Acute Inhalation Toxicity  -  Rat (Harmonized Guideline 870.1300; MRID No. 495680-05). In an acute inhalation toxicity study, groups of young adult Sprague-Dawley rats (5/sex/group) were exposed nose-only to Howler Technical containing 100% Pseudomonas chlororaphis strain AFS009 aerosolized for 4 hours at a concentration of 5.04 mg/L. The animals were observed for 14 days. All animals survived the study. Labored breathing was seen in all animals at one hour after the exposure period with recovery by day 1 in most animals. Labored breathing persisted in only one male after one hour and lasted until day 6. One animal exhibited hypoactivity for one hour post removal. On days 7-14, all animals were reported as active and healthy. All animals lost weight from the exposure but gained it back or surpassed their starting weight by days 3-7. At day 14, all animals showed normal weight gain. No observed abnormalities were noted at necropsy. The inhalation median lethal concentration (LC50), which is a statistically derived concentration that can be expected to cause death in 50% of test animals, for both male and female rats was greater than 5.04 mg/L (Toxicity Category IV). (Refs. 1 and 2). 

It is generally observed that labored breathing, hunched posture, and hypoactivity are signs of receiving a dose by the inhalation route, especially if the dosing rate is at or near the limit dose (2-5 mg/L). There is also consideration of whether the clinical signs are reversible as this indicates recovery from any dosing effect. It is noted that mortality and morbidity are the sole endpoints for the proper assignment of toxicity category for this test. The most credible explanation for the labored breathing and hypoactivity seen in this acute inhalation toxicity test was a response to high concentrations of dosing material. These clinical signs are not considered indicative of a systemic toxicity due to the bacterial agent but due to a high concentration of test material in the test chamber (Ref. 2.)  

4. Acute Pulmonary Toxicity/Pathogenicity and Acute Injection Toxicity/Pathogenicity (Harmonized Guidelines 885.3150 and 885.3200; MRID No. 495680-16). No acute pulmonary toxicity/pathogenicity or acute injection toxicity/pathogenicity studies were submitted; instead, a scientific rationale was submitted requesting waiver of these data requirements. The Agency determined that these data were not necessary and waived the requirements for these data based on the following: (1) the lack of evidence of adverse effects in the acute oral toxicity/pathogenicity study and the acute oral toxicity study; (2) the transience of the impacts in the acute inhalation toxicity study; and (3) the knowledge that the microbe grows best below 86°F (30°C) and in the presence of oxygen. Based on this information, EPA was able to assess the potential of Pseudomonas chlororaphis strain AFS009 to cause infection or pathogenicity by the pulmonary or injection routes of exposure. The temperature (below 30[o]C) and oxygen growth limitations of this microbe are not expected to lead to infection during an intravenous exposure study. There was adequate evidence from an acute oral toxicity/pathogenicity study that the microbe was not infectious. Natural exposures through food to Gram-negative microbes in general are already widespread and harmless if the microbes are not pathogens. Pseudomonas chlororaphis strain AFS009 is not identified as a mammalian pathogen in the scientific literature, and this status is supported by the acute oral toxicity/pathogenicity test results and a literature review. The results of the acceptable oral toxicity/pathogenicity study with a demonstrated pattern of clearance address the injection route of exposure and support waiving the acute injection toxicity/pathogenicity study. The results of the acute inhalation toxicity study with a 4-hour exposure and no mortalities address the inhalation endpoint and support waiving the acute pulmonary toxicity/pathogenicity study. (Ref. 1).

5. Acute Dermal Toxicity  -  Rat (Harmonized Guideline 870.1200; MRID No. 495680-04). An acceptable acute dermal toxicity study demonstrated that Pseudomonas chlororaphis strain AFS009 was not toxic to male and female rats when dosed at 5,000 mg/Kg of body weight for 24 hours. The dermal NOAEL for male and female rats was greater than 5,000 mg/Kg body weight (Toxicity Category IV). (Ref. 1).

6. Acute Eye Irritation  -  Rabbit (Harmonized Guideline 870.2400; MRID No. 495680-06). An acceptable acute eye irritation study demonstrated that Pseudomonas chlororaphis strain AFS009 was not irritating to the eye (Toxicity Category IV). (Ref. 1).

7. Primary Dermal Irritation  -  Rabbit (Harmonized Guideline 870.2500; MRID No. 495680-07). An acceptable primary dermal irritation study demonstrated that Pseudomonas chlororaphis strain AFS009 was not dermally irritating (Toxicity Category IV). (Ref. 1).

Based on the lack of toxicity, infectivity, or pathogenicity in any of the available studies, EPA has not identified any toxicological points of departure. Consequently, EPA's aggregate risk assessment of the Pseudomonas chlororaphis strain AFS009 is qualitative, rather than quantitative, in nature.  

III. Aggregate Exposure

In examining aggregate exposure, FFDCA section 408 directs EPA to consider available information concerning exposures from the pesticide residue in food and all other non-occupational 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: Pseudomonas chlororaphis is naturally found in agricultural settings (i.e., in water, in soils and on plants), and use of Pseudomonas chlororaphis strain AFS009 as a pesticide on food is expected to result in residues in or on food. When consumers wash their produce, it is anticipated that levels of Pseudomonas chlororaphis strain AFS009 and its associated metabolites may be reduced. 
Drinking Water Exposure: Since Pseudomonas chlororaphis is naturally present in soils and on plants, exposure to Pseudomonas chlororaphis from surface water and possibly ground water can be expected. Moreover, use of Pseudomonas chlororaphis strain AFS009 as a pesticide on food is expected to result in residues in or on drinking water. Water treatment processes should remove any Pseudomonas chlororaphis strain AFS009 present. Should this microbial pesticide be present, no adverse effects are expected from exposure to Pseudomonas chlororaphis strain AFS009 through drinking water, based on the results of the toxicological studies. 
Other Non-Occupational Exposure: Since the Pseudomonas chlororaphis strain AFS009 end-use products allow for applications in residential settings, including to bedding plants, annuals and perennials, home gardens, and ornamental trees and shrubs, exposure to the bacterium from its pesticide use would be likely. Pseudomonads, however, are already present in soil and on plant roots, and exposures to Pseudomonas chlororaphis strain AFS009 from pesticidal applications do not present a risk concern, particularly in light of available data that demonstrate it is not toxic or irritating and is not likely to be infective or pathogenic: 

 Dermal exposure. Pseudomonas chlororaphis strain AFS009 was shown to be non-toxic and is not irritating to the skin (acute dermal toxicity and primary dermal irritation data).
 Inhalation exposure. Pseudomonas chlororaphis strain AFS009 was shown to be non-toxic and is not likely to be pathogenic or infective. Initial high doses to the lungs caused transitory effects that cleared within all animals except one by 24 hours. These transitory clinical signs are not considered indicative of a systemic toxicity due to the bacterial agent but due to a high concentration of test material in the test chamber (Ref. 2). Inhalation of large quantities of Pseudomonas chlororaphis strain AFS009 is not expected for homeowner applications because a concentrated product is not used for purposes other than manufacture and repeated exposure of high doses of concentrated product in home and garden sites is not anticipated.

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, EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."

Pseudomonas chlororaphis strain AFS009 is not toxic and does not have a common mechanism of toxicity with other substances. Consequently, FFDCA section 408(b)(2)(D)(v) does not apply.

V. Determination of Safety for the U.S. Population, Infants and Children

A. U.S. Population

For all of the reasons discussed previously, EPA concludes that there is reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of Pseudomonas chlororaphis strain AFS009. This includes all anticipated dietary exposures and all other exposures for which there is reliable information.

B. Infants and Children

FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold (10X) margin of 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 EPA determines based on reliable data that a different margin of safety will be safe for infants and children. This additional margin of safety is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, EPA either retains the default value of 10X or uses a different additional safety factor when reliable data available to EPA support the choice of a different factor. 

No endpoints were identified based on the available data for Pseudomonas chlororaphis strain AFS009, and differential effects to infants and children are not expected. Pseudomonads, such as Pseudomonas chlororaphis strain AFS009, are already very commonly present on a wide range of foods, including those consumed raw or with minimal processing. Because there are no threshold levels of concern to infants and children from Pseudomonas chlororaphis strain AFS009, EPA concludes that no additional margin of safety is necessary to protect infants and children.

VI. Conclusion
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 chlororaphis strain AFS009. Therefore, EPA is establishing an exemption from the requirement of a tolerance for residues of Pseudomonas chlororaphis strain AFS009 in or on all food commodities when used in accordance with label directions and good agricultural practices.

VII. References

1. U.S. EPA. 2016. Howler products containing Pseudomonas chlororaphis strain AFS009. Memorandum from J.V. Gagliardi, Ph.D. through J.L. Kough, Ph.D. to S. Cerrelli dated November 15, 2016 (available as a "Supporting Document" within Docket ID Number EPA-HQ-OPP-2016-0251 at http://www.regulations.gov).

2. U.S. EPA. 2017. Comments on Acute Inhalation Toxicity Study (870.1300) for Microbial Pesticide Technical Pseudomonas chlororaphis AFS 009. Memorandum from J.L. Kough, Ph.D. and M. Perry to M. Mendelsohn dated May 9, 2017 (available as a "Supporting Document" within Docket ID Number EPA-HQ-OPP-2016-0251 at http://www.regulations.gov).

3. Khan, A., J.C. Sutton and B. Grodzinski. 2003. Effects of Pseudomonas chlororaphis on Pythium aphanidermatum and Root Rot in Peppers Grown in Small-scale Hydroponic Troughs. Biocontrol Science and Technology 13(6):615-630.

4. U.S. EPA. 2014. Chapter 7 of the Label Review Manual (Precautionary Statements) (Revised July 2014). Available from https://www.epa.gov/sites/production/files/2015-03/documents/chap-07-jul-2014.pdf.

5. Shen, X., M. Chen, H. Hu, W. Wang, H. Peng, P. Xu and X. Zhang. 2012. Genome Sequence of Pseudomonas chlororaphis GP72, a Root-Colonizing Biocontrol Strain. Journal of Bacteriology 194(5):1269-1270.

6. Tombolini, R., D.J. van der Gaag, B. Gerhardson and J.K. Jansson. 1999. Colonization Pattern of the Biocontrol Strain Pseudomonas chlororaphis MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein. Applied and Environmental Microbiology 65(8):3674-3680.

