UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM 

## AUG 2 9 2012

SUBJECT:

FROM:

TO: Review ofArysta Life Science's Analysis ofKasugamycin's Safety with Regard to Its Microbiological Effect on Bacteria ofHuman Health Concern (FDA/CVM Guidance to Industry # 152)
Karen P. Hicks, Microbiologist (CTT Leader) Product Science Branch Antimicrobials Division (751 OP)
John L. Kough, Ph.D., Biologist Microbial Pesticides Branch, B · pes · cides and Pollution Prevention Division ( 511 ) Mary Waller (RM21) Shaunta Hill Fungicide Branch Registration Division (RD, 7505P)

ACTION REQUESTED: To review information related to the potential ofkasugamycin's agricultural use to select for bacteria ofhuman health concern with enhanced resistance to clinical antibiotics. This analysis is adapted from the Food and Drug Administration's Center for Veterinary Medicine's Guidance to Industry # 152 which was designed to address the microbial resistance associated with new animal drug uses.
DATA REVIEW RECORD
Active Ingredient: Product Name: Company Name: Title ofReport:
Authors: Kasugamycin Kasumin2L Arysta LifeScience North America LLC Kasugamycin: Benefits Overview and Resistance Potentia l Evaluation (per FDA Guidance Document 152) Gary R. Orr, Martin Wigglesworth

1Date: 

### Report No: MRID:
Title ofReport:
Authors: Date: April 13, 2011 A

RY 413

-245565 484559-01 Monitoring for Antibiotic Resistance in Apple Orchards Treated with Kasugamycin George W. Sundin, Gayle C. McGhee, Michigan State University December 1, 2011

BACKGROUND : In 2010, Arysta Life Science ofNorth America petitioned EPA (the Agency) for the registration (under FIFRA Section 3) ofthe aminoglycoside antibiotic kasugamycin for control ofbacterial diseases on tomatoes, peppers, walnuts and pome fruits (apples and pears). Prior to the registration request, Arysta petitioned for and received an import tolerance for kasugamycin on Crop Group 8 (Fruiting Vegetables except cucurbits ). The import tolerance was granted to Arysta at 0.04ppm in 2005. In addition to the import tolerance, kasugamycin has been approved under a FIFRA Section 18 eme{gency exemption with a temporary tolerance for residues less than 0.05ppm resulting from use on apples in Michigan to control streptomycin resistant strains ofthe fire blight bacterium (Erwinia amylovora). Section 18s have been authorized yearly for kasugamycin use in Michigan since 2009. An analysis ofthe resistance development issue was included in t he section 18 approval (memorandum from W.T. Drew, M. Dow, and 

### L. Hansen to A. Ertman and D. Rosenblatt, 29 April2009). In addition to the toxicology and environmental effects data for the current section 3 registration request, Arysta was asked to provide an analysis ofthe microbiological effects on bacteria ofhuman health concern associated with the proposed expanded uses. This analysis was requested due to issues associated with antibiotic use in agriculture.
DISCUSSION:
The qualitative analysis EPA conducted herein is based on the FDA ' s Guidance to Industry 152 with modifications from PRN 2001

-5 appropriate to managing resistance in agricultural chemicals. The adaptation ofthe FDA's 152 Guidance to Industry for the assessment of agricultural use of antibiotics is useful in providing consistent public health goals for deploying antibiotics outside clinical medicine. There are, however, significantly different considerat ions between the animal husbandry uses for which the FDA guidance was developed and agricultural antibiotic use scenarios which will be highlighted in the analysis.
The qualitative risk assessment consists of a release assessment, an exposure assessment, and a consequence assessment. These three elements provide the information to make an overall qualitative risk estimate.
The release assessment considers the probability that resistant bacteria are present on food commodities as a result of antibiotic use on crops. In the case of an agricultural use ofan antibiotic

2on a food crop, the primary expression ofthis resistance would be a failure ofthe antibiotic to control the intended disease. Another adverse outcome could be the appearance ofresistance to epiphytic or soil bacteria to the antibiotic in question. Either ofthese scenarios could indicate an increase in the likelihood of antibiotic resistance transfer to microbes ofhuman health concern and require a closer examination ofthe resistance trait to determine its mobility among bacterial populations.
The exposure assessment considers the probability that humans would ingest or be exposed to bacteria from the treated food commodity. In the case of an agricultural use antibiotic, the incidence offoodborne illness associated with consumption ofthe crop would provide the background for the exposure scenarios. Another consideration would be increasing exposure to bacteria ofhuman health concern through the use of improperly treated animal manures. The consequence assessment considers the probability that the exposure to resistant bacteria could result in an adverse health consequence. Any change in the incidence offoodborne disease found with consumption ofthe treated crop or an increased failure to treat human disease related to ingesting the crop would be pertinent for the consequence assessment.
Given the importance of antibiotics to control bacterial diseases in humans, domestic animals and plants, it is essential that procedures to lessen the likelihood that antibiotic use in crops leads to selection for resistant bacterial populations be adopted. The guidance given in PRN 2001-5 outline general considerations for prudent agricultural use including the avoidance of single chemistry for control, rotating control measures over the season, employing tank mixes to enhance the selection pressure ofthe sprays used, and basing control strategies on integrated pest management programs. An important first step in any resistance control scheme is to monitor for loss offield efficacy and confirm its source. EPA assumes that loss of field efficacy for the target bacterium, while not the sole indication of antibiotic resistance of importance to bacteria ofhuman health concern, is an important sign that continued agricultural use could affect clinical medicine.
There is the potential for selection for resistance to kasugamycin in phytopathogens and environmental bacteria will occur with its broader adoption as a control measure for bacterial plant diseases. There are currently no clinical uses for kasugamycin and a low likelihood that kasugamycin will have cross resistance for other antibiotics based on mechanistic considerations
. However ,knowledge ofthe connection between kasugamycin resistance and possible cross resistance to clinically important antibiotics is lacking. Additionally , The effect ofkasugamycin resistance in environmental bacteria and the effect of transfer ofthis resistance to bacteria ofhuman health concern are unknown

. Thus, to reduce the likelihood ofbacteria developing resistance to kasugamycin, sound resistance management practices and a monitoring program should be employed when using this product.
RECOMMENDATION: The following parameters are recommended for the proposed use patterns ofkasugamycin.
1. Labeling:



### 3• Respective to the end-use product (Kasumin 2L), the label should be amended to include the known mode of action "MOA" infonnation for kasugamycin. The placement and detail ofthe MOA infonnation should follow the Agency's Pesticide Registration Notice (PRN) 200 1-5. • Kasugamycin should be used in rotation and/or tank mixes with other EPA approved bactericides (ex. Coppers).
• As specified under the Section 18 approvals for kasugamycin, the following restrictions should be included o Kasugamycin may not be applied as the first spray ofthe season for all proposed uses. It may only be applied after the first spray ofregistered alternatives. o The number of consecutive applications ofkasugamycin is limited to two for all proposed uses
. o Kasugamycin may not be used in orchards in which the soil has been fertilized with untreated animal manures. o Only the specified application rates can be used.

• The proposed label for Kasumin 2L currently includes the following information. ''Resistance Management: To reduce the likelihood of bacteria developing resistance to kasugamycin, sound resistance managementpractices should be employed when using thisproduct. Suchpractices include limiting the consecutive application of Kasumin 2L, and alternating Kasumin 2L applications w ith other bactericides that have a different mode of action. "
This section should be revised to reflect the following. "Resistance Management: Kasumin 2L (kasugamycin) is a Group 24 fungicide. The mode of action for Kasumin 2L is the inhibition of protein synthesis. To reduce the likelihood of bacteria developing resistance to kasugamycin,follow the crop specific resistance management and use direction informationpresent on this labeling. Use of thisproduct should also conform to resistance managementpractices/strategies established for the crop and use area, (for example the use of IP 

### M, resistant crop varieties, etc.)

. Consultyour local extension/crop consultant or State agricultural authority 

### if reduced efficacy is suspected. "
2. Monitoring Program: The existing data generated for the Section 18 on apples in Michigan supports a "low" overall qualitative risk estimation for the first three years ofuse. Implementing a monitoring plan similar to that done in Michigan for the other growing regions of apple and pear would provide a baseline ofkasugamycin resi stance in these other regions. Use ofkasugamycin in walnuts, · tomatoes and peppers should also be contingent on establishing baseline data on kasugamycin resistance and the presence ofbacteria ofhuman health concern on all the treated crops in each crop production region. The results ofthe monitoring plans should be reported to t he Agency on an annual basis and may continue for three years. Should the Agency determine the

4monitoring results indicate additional concern; the monitoring plan may be extended.

SUMMARY OF DATA SUBMITTED: Arysta provided a summary ofthe benefits ofthe use of kasugamycin along with the 152 analysis. The benefits information is not reviewed 

### in this memorandum. The submission is divided into four sections that correlate with the format proposed by the 152 Guidance to Industry: 1) The Release Assessment which covers antibiotic product characterization information, use pattern and a discussion ofwhat is known for target susceptibility, spectrum ofactivity, resistance mechanisms and selection pressure. 2) The Exposure Assessment which examines the human exposure to food-borne bacteria by way ofthe treated crop. 3) The Consequence Assessment which relates the foregoing information to the loss ofefficacy of antibiotics for human clinical use. 4) The Risk Estimation integrates the release, exposure and consequence findings with an overall estimate ofrisk (ranked low, medium or high); associated with the proposed antibiotic use. Most ofthe differences between the animal uses and the agricultural use relate to the lack ofpharmacodynamics of antibiotics for crop use, the lower likelihood of direct contamination offood crops by bacteria ofhuman health concern and the much broader exposure of sprayed antibiotics in agriculture compared to injected or orally consumed antibiotics in animal husbandry. These differences will be reiterated in the individual section discussions.
RELEASE ASSESSMENT The boundaries ofthe release assessment relate to the proposed use pattern on apple and pear, tomato, pepper and walnut. The acres treated were estimated for each crop with apple and pear being 314,000 acres to control fire blight; 387, 000 acres oftomato for bacterial spot and speck ; 279,000 acres ofpepper for bacterial spot and 208 ,000 acres ofwalnut for bacterial blight. These numbers , representing an upper limit of 100% ofthe affected crop treated with antibiotic; will vary annually according to disease pressure driven by weather conditions , and are the most recent available figures developed by the company.
The proposed label allows a maximum of 3 applications per year with a one day pre-harvest interval for tomatoes and peppers. For apples and pears, a maximum of4 applications per year with a 30 day pre-harvest interval are allow. Four applications per year with a 100 day pre-harvest interval are proposed for walnuts. The application technology for these uses is by conventional ground application which would most likely be by boom or handheld sprayers for the fruiting vegetables and by air blast for the orchard trees. The directions for use indicate that this product should be used when conditions favor disease development.
The conditions favoring disease development vary with each host

-pathogen combination. For fire blight, the disease is first found in blossoms in the spring and is favored when temperatures are around 80°F with heavy rain, dew and mists. The bacterium prefers flowers and succulent fast growing tissues (emerging leaves and expanding stems). Secondary infections can continue throughout the growing season but new infections are reduced in the presence ofhot dry weather. Economic damage is due to fruit loss, tree disfigurement and mortality. Walnut blight is favored by free moisture and is not as affected by temperature as fire blight. The bacterium is harbored 

### in

5dormant buds and infected twigs and primary infection occurs as buds break and catkins emerge. The economic damage results from nut infection and quality loss. For bacterial speck oftomato, the disease develops early in the season when low temperatures and heavy rainfall occur. The bacterial spot disease oftomatoes and peppers is favored by warm, humid weather and heavy precipitation. Both bacterial speck and spot cause economic damage by defoliation and fruit quality loss. There are computer assisted disease forecasting systems for both fire blight ofpome fruit and bacterial blight ofwalnut but no similar system exists for the bacterial diseases oftomato and pepper. Kasugamycin is in the aminoglycoside class of antibiotics but differs from other clinically important members in the class. Kasugamycin acts against protein synthesis by inhibiting the formation ofthe initiation complex oftypical or canonical mRNA with 30S ribosomal subunits. Several classes of organisms can also translate mRNA without this canonical leader by binding 70S ribosomal subunits directly. Therefore, resistance to kasugamycin can occur in some populations at a rather high frequency. Clinically important aminoglycosides also act on protein syn thesis but act further in the process at the level ofmiscoding ofthe mRNA within the 70S complex. A possible mechanism for cross resistance involved with both kasugamycin and the other clinically important aminoglycosides has not been reported. While the mode of action described above is bactericidal, the original use ofkasugamycin was for control of a fungal disease ofrice. The antifungal mode of action has not been discussed in the Arysta submission.
The spectrum of activity for kasugamycin is gram negative bacteria and a limited number offungi (Pyricularia oryzae, Colletotrichum lagenarium and Cladosporium fulvum have been mentioned). Although kasugamycin is used in Japan for control ofrice blast (Pyricularia oryzae ), there are currently no fungal label claims or requests for including control of any fungal diseases in this submission. Arysta presented results from screening kasugamycin for activity against a set of clinically important bacteria and the related activity levels for the clinically important aminoglycosides. The levels reported for kasugamycin to have activity against clinically important bacteria were typically 2 orders ofmagnitude higher than that reported for the other aminoglycosides and kasugamycin is said by Arysta to be not effective at practical use levels. The bacterial species tested were Micrococcus luteus, Bacillus subtilis, B. anthracis, Corynebacterium bovis, Escherichia coli , Shigella dynsenteriae, Salmonella typhi, Pseudomonas aeruginosa, P

. fluorescens, P. maltophila and Yersina entercolitica. As previously mentioned, kasugamycin showed no activity against the gram positive bacteria Micrococcus luteus, Bacillus subtilis, and B

. anthracis. The results were from a paper by T. Tamamura and K. Sato in the Japanese Journal of Antibiotics 52:57

- 67 (1999).
To address the cross resi stance to other aminoglycoside antibiotics, Arysta presented data on the sensitivity oftarget plant pathogenic bacteriaXanthomonas campestris and Pseudomonas syringae. These st rains were selected for kasugamycin resistance in the laboratory then examined for sensitivity to streptomycin and kanamycin. The kasugamycin resistant strains showed similar levels of sensitivity to streptomycin and kanamycin as that ofthe kasugamycin sensitive strains. This is purported to indicate a lack ofcross resistance to other aminoglycosides when kasugamycin resistance is found.

6The pharmacodynamics and pharmacokinetics ofkasugamycin were not discussed for the plant uses. Since there is little indication that externally applied antibiotics are taken up and distributed

within plants, the endpoint ofpharmacodynamics and pharmacokinetics is not relevant. The most analogous endpoint would be environmental persistence ofthe antibiotic

. There was a claim by Arysta that kasugamycin has a half-life in aerobic soil of about 40 days. The current EFED assessment indicates t hat kasugamycin half lives in various natural environments range from 5.7 days to 12.3 days in field conditions and 17.4 to 45 days in lake water or lake or river water and sediments. The longest half lives in a natural environment were in anaerobic environments of 105 days in an anaerobic aquatic environment and 147 days in an anaerobic paddy soil en vironment. The resistance mechanisms were not discussed but it is known that kasugamycin resistance can be selected in the laboratory and that, in some cases, this resistance is due to structural changes in the 16S ribosomal subunit (R.A. Zimmerman 

### et al. , 197 3). In 

### Escherichia coli, this type ofresistance is due to an enzyme with ribosome methylation activity. The rate ofthis mutation to kasugamycin resistance and its occurrence in target plant pathogenic bacteria are not known. There is anecdotal evidence that kasugamycin resistance is related to overuse according to the company. It is not clear ifthis is referring to the fungicidal or bactericidal use ofkasugamycin. The company also indicated that this resistance was resolved by rotation to another control measure resulting in the reappearance of a sensitive population.
The baseline level ofresistance to kasugamycin in environmental bacteria and tracking the effect of kasugamycin treatments on the frequency ofresistance to clinically important classes of antibiotics in these environmental isolates was the intent of a report done by Gayle C. McGhee and George W. Sundin at Michigan State University in conjunction with the section 18 in Michigan for fire blight on apple. The clinical antibiotics were cefotaxime , ampicillin, tetracycline , gentamicin and streptomycin. The design ofthe study looked at bacterial populations in both kasugamycin treated and untreated apple orchards. Bacterial populations ofthe apple leaf and the soil environment under the apple trees were examined in both kasugamycin treatment situations. The results indicate higher numbers ofbacteria (usually at least two orders ofmagnitude) being present in the soil than on apple leaftissue. The level ofresistance to the surveyed clinical antibiotics was low in populations ofepiphytic bacteria recovered from apple leaves

. Higher frequencies ofresistance to the surveyed antibiotics and greater species diversity ofbacterial populations were found in soil samples. The percentage ofbacteria resistant to the surveyed antibiotics was always higher in soil populations ofbacteria compared to foliar isolates except for streptomycin. This higher population of streptomycin resistant bacteria from foliar samples may be due to the use of streptomycin to control fue blight. The data from the 2011 survey did not reveal any significantly enhanced selection for resistance to the surveyed antibiotics relat ed to the kasugamycin treatment.
PARAMETER COMMENT RELEASE RATING Mechanism of activity Different site from other Low aminoglycosides ; low likelihood to have cross res istance for other antibiotics. Spectrum of activity Gram negatives ; less effective Low against human pathogeni c species.



### 7Resistance mechanism Ribosomal methylating enzyme Medium reported; others possible. Selection pressure Wide use likely to create Medium significant selection pressure Resistance transfer No data available; likely to be Low? easily transferred since related to

enzyme activity, not ribosomal structural sequence. The release assessment rating for the proposed uses would be expected to be " low" based on the limited information available for kasugamycin. Since there is no definitive da ta to cite for the resistance transfer endpoint, this would be an area for further examination and a possible monitoring endpoint for the expanded uses.
EXPOSURE ASSESSMENT
The adaptation ofthe FDA's 152 guidance from the veterinary to the agricultural scenario has two aspects that great ly complicate the exposure assessment. The animal drug use, administered directly to animals in medicated feeds or by injection, has much less environmental exposure compared to agricultural sprays of antibiotics. The animal use is more likely to have direct effects on potential antibiotic resistance in human pathogens due to the presence ofbacteria ofhuman health concern in the intestinal flora oftreated animals. The intestinal tract oftreated animals also provides high density, homogenous bacterial populations for genetic interactions with consistent temperatures and environmental conditions. The agricultural use has a much greater environmental exposure due to the application by air blast or other spray technologies. The agricultural use exposes more different types ofbacteria to select ion for antibiotic resistance but may have less direct impact on bacteria ofhuman health concern since they are a relatively minor component of the general environment (unless improperly handled animal manures are used in treated fields).
The human health concern comes mostly from contamination offood crops with bacteria ofhuman health concern and the transfer ofresistance traits to these microbes ofhuman health concern, not the environmental strains with resistance traits. Establishing a link between kasugamycin resistance and resistance to clinically important antibiotics (especially other aminoglycosides), and quantifying the potential for resistance traits to move from environmenta l bacteria into clinica lly important bacteria should be a focus ofinformation generated for the exposure assessments.
The current exposure assessment is based on information collected for the section 18 use in Michigan on apple orchards. The exposure assessment for apples in Michigan is not expected to be significantly different from that reported in 2009 memorandum. Since the proposed use expands to all pome fruit growing areas, the amount ofthe food commodity being consumed will be higher. For the uses on walnuts , tomato and pepper, the amount offood commodity being consumed is "high". The level of food commodity contamination for all the crops on the proposed section 3 label is low but variable. The incidence oftomato, pepper and apple being implicated in food poisoning incidents has increased in recent years. This would yield a rating of " medium" for the exposure assessment.
8Arysta has requested new crops that will expand the proposed use sites to new geographical areas where the current exposure assessment is probably not valid. The label directions for use on tomato and pepper indicate that there will potentially be kasugamycin sprays later in the growing season and with a shorter post harvest interval than for the orchard tree uses
. To account for the increased exposure from expanded uses, Arysta should provide information on existing resistance in plant associated and soil bacteria, similar to that done in Michigan for the section 18 , in the geographical regions where kasugamycin is expected to be used on walnut, pepper and tomato. The exposure assessment could also be refined for the larger area of apple and pear culture to be treated outside of Michigan. CONSEQUENCE ASSESSMENT
The ranking for the consequence assessment within the 152 guidance is expressed as " importan t", "highly important" and " critical". The consequence assessment for kasugamycin is "important" for the first three years of use. " Important" is the lowest risk ranking for FDA ' s 152 consequence assessment. This low risk rating is due to the submitted data from the limited use in Michigan showing no change in resistance frequency to other aminoglycosides in the presence of kasugamycin resistance in tested bacteria. This can also be supported by the difference in sensitivity to kasugamycin for plant pathogenic bacteria compared to clinically important species. There are currently no clinical uses for kasugamycin. This assessment may change to "highly important" or "critical" if it is found that kasugamycin resistance, when it develops, can provide cross resistance to clinically important aminoglycosides. The data to establish or refute this supposit ion is not available. It is unlikely there is cross resistance at the current time due to kasugamycin's activity at a different binding site in bacterial protein translation and the limited use under a section 18 in Michigan. Currently, kasugamycin has only been used as an emergency option under section 18 in the United States. The use has been limited by both time and location for only a single crop, apples. The section 3 registration request expands the use to include other pome fruits, tomato and pepper, and walnut so the exposure, and consequently, the select ion pressure for resistance, will expand significantly. In the anecdotal case ofkasugamycin resistance mentioned but not detailed in the submission, the company stated that with proper rotation ofother alternative pesticides, the resistance to kasugarnycin was resolved. Arysta claims it will institute a product stewardship program, provide label language to limit use to no more than 4 times per season, encourage rotation with other non-antibiotic agents and monitor use sites for the appearance ofkasugamycin resistant microbes.
OVERALL QUALITATIVE RISK ESTIMATION
The risk estimation integrates the three previous assessments to provide an overall rating. There are certain uncertainties associated with these assessments. For the release assessment, it was noted that information is lacking on kasugamycin susceptibility for the range ofbacteria associated with food borne incidents in the crops proposed to be treated and the effect ofkasugamycin to co-select



### 9for resistance to other clinical antibiotics. For the exposure assessment, data on the actual level of contamination with bacteria ofhuman concern on the food commodities proposed for use is not available. For the consequence assessment, it is clear that there are currently no clinical uses for kasugamycin but ifthe agricultural use did co-select for resistance to clinically important antibiotics, the rating could change.
Given the "low" release, "medium" exposure and "important" consequence ratings, the overall qualitative risk estimation would be "low" for the first three years ofuse in pome fruit, tomatoes, peppers and walnuts. This finding may change after considering the monitoring results and additional information.
REFERENCES Tamamura, T. and Sato, K. (1999) Comparative studies on in vitro activities ofkasugamycin and clinically used aminoglycoside antibiotics, Japanese Journal ofAntibiotics 52:57-67. Zimmermann, R.A., Ikeya, Y. and P.F. Sparling (1973) Alteration ofRibosomal Protein S4 by Mutation Linked to Kasugamycin Resistance in Escherichia coli, Proceedings ofthe National Academies of Science 70:71-75.

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