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 of Arysta Life Science's Analysis ofKasugamycin's Safety with Regard 

to Its Microbiological Effect on Bacteria of Human 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 of human 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 of Report: 

Authors: 

Kasugamycin 

Kasumin2L 

Arysta LifeScience North America LLC 

Kasugamycin: Benefits Overview and Resistance Potential Evaluation (per 

FDA Guidance Document 152) 

Gary R. Orr, Martin Wigglesworth 

1 
Date: 

Report No: 

MRID: 

Title of Report: 

Authors: 

Date: 

April 13, 2011 

ARY 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) of the aminoglycoside antibiotic kasugamycin for 

control of bacterial 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 

of the fire blight bacterium (Erwinia amylovora). Section 18s have been authorized yearly for 

kasugamycin use in Michigan since 2009. An analysis of the resistance development issue was 

included in the 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 of the 

microbiological effects on bacteria of human 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 of the 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 considerations 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 of an antibiotic 

2 
on a food crop, the primary expression of this resistance would be a failure of the antibiotic to 

control the intended disease. 

Another adverse outcome could be the appearance of resistance to epiphytic or soil bacteria to the 

antibiotic in question. Either of these scenarios could indicate an increase in the likelihood of 

antibiotic resistance transfer to microbes of human health concern and require a closer examination 

of the 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 of food borne illness associated with consumption of the crop would provide the 

background for the exposure scenarios. Another consideration would be increasing exposure to 

bacteria of human 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 of food borne disease found 

with consumption of the 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 of the 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 of field 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 of human 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 

of the 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 of this resistance to bacteria of human health 

concern are unknown. Thus, to reduce the likelihood of bacteria 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 of kasugamycin. 

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 of the MOA infonnation should follow the Agency's Pesticide 

Registration Notice (PRN) 2001-5. 

• 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 of the season for all 

proposed uses. It may only be applied after the first spray of registered 

alternatives. 

o The number of consecutive applications of kasugamycin 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 management practices should be employed when 

using this product. Such practices include limiting the consecutive application 

of Kasumin 2L, and alternating Kasumin 2L applications with 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 information present 

on this labeling. Use of this product should also conform to resistance 

management practices/strategies established for the crop and use area, (for 

example the use of IP M, resistant crop varieties, etc.). Consult your 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 of use. Implementing a monitoring plan 

similar to that done in Michigan for the other growing regions of apple and pear would provide 

a baseline ofkasugamycin resistance 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 of bacteria ofhuman health concern on all the treated crops in each 

crop production region. The results of the monitoring plans should be reported to the Agency 

on an annual basis and may continue for three years. Should the Agency determine the 

4 
monitoring 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 of what is known for target susceptibility, 

spectrum of activity, resistance mechanisms and selection pressure. 2) The Exposure Assessment 

which examines the human exposure to food-borne bacteria by way of the treated crop. 3) The 

Consequence Assessment which relates the foregoing information to the loss of efficacy of 

antibiotics for human clinical use. 4) The Risk Estimation integrates the release, exposure and 

consequence findings with an overall estimate of risk (ranked low, medium or high); associated 

with the proposed antibiotic use. Most of the differences between the animal uses and the 

agricultural use relate to the lack of pharmacodynamics of antibiotics for crop use, the lower 

likelihood of direct contamination of food crops by bacteria of human 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 of the 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 of tomato for bacterial spot and speck; 

279,000 acres of pepper for bacterial spot and 208,000 acres of walnut for bacterial blight. These 

numbers, representing an upper limit of 100% of the 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 of 4 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 of hot 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 

5 
dormant 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 of tomato, the 

disease develops early in the season when low temperatures and heavy rainfall occur. The bacterial 

spot disease of tomatoes 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 of pome fruit and bacterial 

blight of walnut but no similar system exists for the bacterial diseases of tomato 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 of the 

initiation complex of typical 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 synthesis but act further 

in the process at the level of miscoding of the 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 of kasugamycin was for control of a fungal disease of rice. 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 of fungi 

(Pyricularia oryzae, Colletotrichum lagenarium and Cladosporium fulvum have been mentioned). 

Although kasugamycin is used in Japan for control of rice 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 of magnitude 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 resistance to other aminoglycoside antibiotics, Arysta presented data on the 

sensitivity of target plant pathogenic bacteriaXanthomonas campestris and Pseudomonas syringae. 

These strains 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 of the kasugamycin sensitive strains. This is 

purported to indicate a lack of cross resistance to other aminoglycosides when kasugamycin 

resistance is found. 

6 
The pharmacodynamics and pharmacokinetics of kasugamycin were not discussed for the plant 

uses. Since there is little indication that externally applied antibiotics are taken up and distributedwithin plants, the endpoint of pharmacodynamics and pharmacokinetics is not relevant. The most 

analogous endpoint would be environmental persistence of the 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 that 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 14 7 days in an anaerobic paddy soil environment. 

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. , 1973). In Escherichia coli, this type of resistance 

is due to an enzyme with ribosome methylation activity. The rate of this 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 

if this is referring to the fungicidal or bactericidal use of kasugamycin. 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 of resistance to kasugamycin in environmental bacteria and tracking the effect of 

kasugamycin treatments on the frequency of resistance 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 of the study looked at bacterial populations in both kasugamycin treated 

and untreated apple orchards. Bacterial populations of the apple leaf and the soil environment 

under the apple trees were examined in both kasugamycin treatment situations. The results indicate 

higher numbers of bacteria (usually at least two orders of magnitude) being present in the soil than 

on apple leaf tissue. The level of resistance to the surveyed clinical antibiotics was low in 

populations of epiphytic bacteria recovered from apple leaves. Higher frequencies of resistance to 

the surveyed antibiotics and greater species diversity of bacterial populations were found in soil 

samples. The percentage of bacteria resistant to the surveyed antibiotics was always higher in soil 

populations of bacteria 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 related to the kasugamycin treatment. 

PARAMETER COMMENT RELEASE RATING 

Mechanism of activity Different site from other Low 

aminoglycosides; low likelihood 

to have cross resistance for other 

antibiotics. 

Spectrum of activity Gram negatives; less effective Low 

against human pathogenic species. 

7 
Resistance 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 data 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 of the FDA's 152 guidance from the veterinary to the agricultural scenario has two 

aspects that greatly 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 of bacteria of human 

health concern in the intestinal flora of treated animals. The intestinal tract of treated 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 of bacteria to selection for antibiotic resistance but may have less 

direct impact on bacteria of human 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 of food crops with bacteria ofhuman 

health concern and the transfer of resistance traits to these microbes of human 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 environmental bacteria into clinically important 

bacteria should be a focus of information 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 of the food commodity being consumed will be higher. 

For the uses on walnuts, tomato and pepper, the amount of food 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 of tomato, 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. 

8 
Arysta 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 "important", 

"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 

supposition 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 selection 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 of other 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 of bacteria associated with 

food borne incidents in the crops proposed to be treated and the effect of kasugamycin to co-select 

9 
for resistance to other clinical antibiotics. For the exposure assessment, data on the actual level of 

contamination with bacteria of human 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 if the 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 of use 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 of kasugamycin and 

clinically used aminoglycoside antibiotics, Japanese Journal of Antibiotics 52:57-67. 

Zimmermann, R.A., Ikeya, Y. and P.F. Sparling (1973) Alteration of Ribosomal Protein S4 by 

Mutation Linked to Kasugamycin Resistance in Escherichia coli, Proceedings of the National 

Academies of Science 70:71-75. 

10