Page 1 of 48 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES MEMORANDUM DATE: 8/ 17/ 2005 SUBJECT: Kasugamycin. Human Health Risk Assessment for Proposed Food Uses of the Fungicide Kasugamycin on Imported Fruiting Vegetables ( Group 8).

Petition Number: 3E6579. PC Code ( Chemical Number): 230001 DP Barcode: D301735 Regulatory Citation: 40CFR § 180.??? ( Not Yet Established) EPA Registration Number: ( Not Registered) Trade Name: Kasumin ® 2L Chemical Class: Aminoglycoside Antibiotic Fungicide Regulatory Action: Establishment of Permanent Tolerances Risk Assessment Type: Single Chemical, No Aggregate FROM: William T. Drew, Chemist/ Risk Assessor Kelly M. Schumacher, Toxicologist Douglas A. Dotson, Dietary Exposure Assessor Registration Action Branch 2 Health Effects Division ( 7509C)

THROUGH: Richard A. Loranger, Branch Senior Scientist Registration Action Branch 2 Health Effects Division ( 7509C)

TO: Mary Waller/ Lana Coppolino, RM Team 21 Fungicide Branch Registration Division ( 7505C) Page 2 of 48 Table of Contents 1.0 Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.0 Ingredient Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.1 Summary of Proposed Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2 Structure and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.3 Physical and Chemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.0 Metabolism Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1 Comparative Metabolic Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2 Nature of the Residue in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.2.1. Description of Primary Crop Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 9 3.2.2 Description of Livestock Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.2.3 Description of Rotational Crop Metabolism . . . . . . . . . . . . . . . . . . . . . . 9 3.3 Environmental Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.4 Tabular Summary of Metabolites and Degradates . . . . . . . . . . . . . . . . . . . . . . . 10 3.5 Toxicity Profile of Major Metabolites and Degradates . . . . . . . . . . . . . . . . . . . 11 3.6 Summary of Residues for Tolerance Expression and Risk Assessment . . . . . . . . 11 4.0 Hazard Characterization/ Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.1 Hazard Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.2 FQPA Hazard Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2.1 Adequacy of the Toxicity Data Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2.2 Evidence of Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2.3 Developmental Toxicity Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 <IP_ADDRESS> Developmental Toxicity Study in Rats . . . . . . . . . . . . . . 20 <IP_ADDRESS> Developmental Toxicity Study in Rabbits . . . . . . . . . . . . 21 4.2.4 Reproductive Toxicity Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.2.5 Additional Information from Literature Sources . . . . . . . . . . . . . . . . . . 22 4.2.6 Pre and/ or Post Natal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 <IP_ADDRESS> Determination of Susceptibility . . . . . . . . . . . . . . . . . . . 22 <IP_ADDRESS> Degree of Concern Analysis and Residual Uncertainties for Pre and/ or Post natal Susceptibility . . . . . . . . . . . . . . . . 23 4.3 Recommendation for a Developmental Neurotoxicity Study . . . . . . . . . . . . . . . 23 4.3.1 Evidence that Supports Requiring a Developmental Neurotoxicity Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.3.2 Evidence that Supports not Requiring for a Developmental Neurotoxicity Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.4 Hazard Identification and Toxicity Endpoint Selection . . . . . . . . . . . . . . . . . . . 23 4.4.1 Acute Reference Dose ( aRfD)  Females ( 13 to 49 Years of Age) . . . . . 23 4.4.2 Acute Reference Dose ( aRfD)  General Population . . . . . . . . . . . . . . . 24 4.4.3 Chronic Reference Dose ( cRfD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.4.4 Incidental Oral Exposure ( Short and Intermediate Term) . . . . . . . . . . . . 26 4.4.5 Dermal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4.4.6 Dermal Exposure ( Short, Intermediate and Long Term) . . . . . . . . . . . . 26 Page 3 of 48 4.4.7 Inhalation Exposure ( Short, Intermediate and Long Term) . . . . . . . . . . 26 4.4.8 Margins of Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.4.9 Recommendation for Aggregate Exposure Risk Assessments . . . . . . . . 27 4.4.10 Classification of Carcinogenic Potential . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.5 Special FQPA Safety Factor . . . . . . . . . . . . . . 28 4.6 Endocrine Disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 5.0 Public Health Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 6.0 Exposure Characterization/ Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 6.1 Dietary Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 6.1.1 Residue Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 6.1.2 Acute and Chronic Dietary Exposure and Risk . . . . . . . . . . . . . . . . . . . 30 6.2 Water Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 6.3 Residential ( Non Occupational) Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . 31 7.0 Aggregate Risk Assessments and Risk Characterization . . . . . . . . . . . . . . . . . . . . . 31 8.0 Cumulative Risk Characterization/ Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 9.0 Occupational Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 10.0 Data Needs and Label Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 10.1 Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 10.2 Residue Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 References: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Page 4 of 48 1.0 Executive Summary Arvesta Corporation, as an agent for Hokko Chemical Industry Company, Limited ( Japan), has proposed ( in PP# 3E6579) the establishment of permanent tolerances for residues of the fungicide kasugamycin, with CAS Registry Number 6980 18 3 and CAS Name 3 O[ 2 amino 4[( carboxyiminomethyl) amino] 2,3,4,6 tetradeoxy   D arabino hexopyranosyl] D chiro inositol, in/ on the agricultural commodities listed below. The proposal is for tolerances on imported commodities without a US registration. Fruiting vegetables ( Crop Group 8) . . . . . . . . . . . . . . . . 0.04 ppm Tomato juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06 ppm Tomato puree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06 ppm Tomato paste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 ppm The nature of the residue in fruiting vegetables is adequately understood, based upon an acceptable study of kasugamycin metabolism in tomato fruit and foliage. Results of the submitted tomato metabolism study indicate that the major metabolic pathway in tomato involves conjugation of the parent compound, conversion to kasugamycinic acid, and subsequent conjugation of kasugamycinic acid. Conversion of kasugamycin to 2 N acetyl kasugamycin and kasuganobiosamine is thought to be a minor metabolic route. The residue of concern ( ROC) in fruiting vegetables ( for both tolerance setting and risk assessment purposes) is the parent compound, kasugamycin per se ( Kasugamycin RAD Team decision, D301735, meeting of 2/ 23/ 2005). There are currently no established Codex, Canadian, or Mexican MRLs for kasugamycin. The toxicology database is adequate for purposes of risk assessment. Kasugamycin exhibits low acute toxicity. The major effects observed across species in multiple dose studies were decreased body weights and body weight gains. Kasugamycin is not mutagenic and no oncogenic potential was noted in the mouse oncogenicity or in the rat combined chronic/ carcinogenicity studies. Based on the overall weight of the evidence, kasugamycin is classified as " not likely to be carcinogenic to humans." There was no evidence that exposure to kasugamycin results in neurotoxicity. The database is complete with respect to pre and postnatal toxicity and shows no evidence of increased qualitative or quantitative susceptibility in the offspring. Based on the hazard and exposure data, HED recommends that the special FQPA safety factor be reduced to 1X and that no database uncertainty factor is needed because there are no/ low concerns and no residual uncertainties with regard to pre and/ or post natal toxicity. Neither dose nor endpoint was selected for acute dietary exposure in the general population ( or in any of the population subgroups) because an acute toxic endpoint was not identified in the submitted studies. The chronic dietary endpoint is based on increased testicular softening and atrophy observed in a combined chronic toxicity/ carcinogenicity study in rats. The NOAEL for this study is 11.3 mg/ kg/ day, resulting in a chronic reference dose ( cRfD) and chronic population adjusted dose ( cPAD) of 0.113 mg/ kg/ day. The chronic dietary assessment was conducted using DEEM FCID and is very conservative in that 100% crop treated and tolerance level residues were used in all commodities. Exposure estimates include the ROC for kasugamycin originating from food only. With no proposed US registration, there is no expectation that kasugamycin residues would occur in surface or ground water sources of drinking water. Since kasugamycin is proposed for use only on imported fruiting vegetable commodities, the sole anticipated exposure route for the US population is via dietary ( food only) exposure. The Tier 1 analysis indicates that chronic dietary exposures to kasugamycin are below HED's level of concern ( 100% of the cPAD). For chronic Page 5 of 48 dietary ( food only) exposure to kasugamycin, the most highly exposed population subgroup is Children ( 1 to 2 years of age), which utilizes less than 1% of the cPAD. Chronic dietary risk to all other subgroups is less than that of Children ( 1 2). With no proposed US registration, kasugamycin is not intended for use in public, residential, or occupational settings; also, there is no expectation that exposure to kasugamycin residues would occur via water consumption. Ergo, risk assessments for water, residential, aggregate, and occupational exposures were not performed. Based on the submitted crop field trial data, the following use pattern would be supported for fruiting vegetables: application of the Kasumin ® 2L formulation as a foliar broadcast spray in a minimum spray volume of 30 gallons of water per acre ( GPA) using ground equipment, with no spray adjuvants, a minimum re treatment interval ( RTI) of 3 days, and a pre harvest interval ( PHI) of 1 day. The submitted field trial data for peppers and tomatoes are adequate to support the proposed uses on imported fruiting vegetables. No residues were detected above the limit of quantitation ( LOQ); therefore the proposed and recommended tolerances are based on the LOQ. Processing studies on tomato, for the processed commodities tomato puree and tomato paste, have been waived. The proposed residue analytical method is adequate for data collection. The method was submitted to the Analytical Chemistry Laboratory of the Biological and Economic Analysis Division ( ACL/ BEAD) for a petition method validation ( PMV) trial ( PMV Request Memo, D313672, William T. Drew, 3/ 1/ 2005) to determine its suitability for tolerance enforcement. The method subsequently failed the PMV trial ( PMV Results Memo, D313673, Patricia G. Schermerhorn, 7/ 13/ 2005). The method should be modified as recommended by ACL. The fact that an adequate analytical method for tolerance enforcement is unavailable does not impact this risk assessment regarding risk characterization. HED has examined the residue chemistry database for kasugamycin and recommends against the establishment of tolerances for residues of this fungicide in/ on imported fruiting vegetables until the data deficiencies listed below ( OPPTS Residue Chemistry Test Guideline 860.1340) have been resolved. 860.1340 Residue Analytical Method ( Plant Commodities) ( 1) The proposed enforcement method should be modified to include a confirmatory analysis method; alternatively, the petitioner may submit an interference study with all pesticides for which tolerances on tomatoes and/ or peppers have been established. ( 2) The method has failed a PMV trial ( PMV Results Memo, D313673, Patricia G. Schermerhorn, 7/ 13/ 2005), and should be modified to include the revisions recommended by ACL. 860.1550 Proposed Tolerances The available crop field trial data support a tolerance for residues of kasugamycin per se in/ on imported fruiting vegetable commodities ( Crop Group 8) at 0.04 ppm, the method's LOQ. The exaggerated rate tomato field trial data ( reflecting a 5X application rate) indicate that separate tolerances for tomato juice, tomato puree and tomato paste are not required. The proposed tolerances should be revised to reflect the tolerance as recommended by HED and the correct commodity definition as specified in Table 1.0, below. The petitioner should submit a revised Section F for PP# 3E6579 to reflect these changes. Page 6 of 48 TABLE 1.0 Tolerance Summary for Kasugamycin.

Commodity Proposed Tolerance ( ppm) Recommended Tolerance ( ppm) Comments/ Correct Commodity Definition Fruiting Vegetables ( Crop Group 8) 0.04 0.04 Vegetable, fruiting, group 8 Tomato Juice 0.06 None Residues are not expected to exceed the tolerance on the raw agricultural commodity ( RAC). Tomato Puree 0.06 Tomato Paste 0.25 2.0 Ingredient Profile Kasugamycin is a low use rate wide spectrum aminoglycoside antibiotic fungicide produced from Streptomyces kasugaensis that is intended for control of bacterial and fungal diseases on pepper and tomato. Kasugamycin has preventative, curative, and systemic activity at low application rates. It is currently used in over 20 countries on various crops including tomato, paprika, eggplant, rice, and potato. Kasugamycin has a mode of action different from that of the other aminoglycoside antibiotics such as streptomycin. Because kasugamycin is active only against phytopathogenic fungi and bacteria, it has never been employed as a human or veterinaryuse antibiotic.

2.1 Summary of Proposed Uses The petitioner has indicated that Arvesta de Mexico is seeking registration of Kasumin ® 2L, a liquid formulation comprised of 2% kasugamycin ( by weight) as the active ingredient ( ai), for use on rice, potato, pepper, and tomato in Mexico. The product is formulated from kasugamycin hydrochloride hydrate ( 2.3%) and contains 2% kasugamycin ( 0.1667 lb ai/ gal) as the free base. Kasugamycin is also formulated as a co active ingredient ( at 5%) along with copper oxychloride ( at 45%, expressed as copper) in a wettable powder ( WP), designated Kasumin Cobre ® . Additionally, the Kasumin ® formulation is a WP containing 8% kasugamycin. Only tomatoes and peppers will be imported into the US from Mexico. The current petition proposes use of kasugamycin in Mexico, where the principal target organisms are bacteria rot ( Erwinia atroseptica) and leaf mold ( Cladosporium fulvum) on tomato, and bacteria spot ( Xanthomonas campestris, pv vesicatoria) on both tomato and pepper. The petitioner is proposing application of kasugamycin to peppers and tomatoes as a foliar broadcast spray with up to 3 applications at a maximum single application rate of 0.018 pound ai per acre ( lb ai/ A), a maximum seasonal application rate of 0.054 lb ai/ A, and a 1 day PHI. Proposed application timing ( as related to plant growth stage), application equipment type, retreatment intervals ( RTIs), spray volumes, and whether tank mix adjuvants would be proposed were not specified. Based on the submitted field trial data, the following use pattern would be supported for fruiting vegetables: application of the Kasumin ® 2L formulation as a foliar broadcast spray in a minimum spray volume of 30 GPA using ground equipment, with no spray adjuvants, a minimum RTI of 3 days, and a PHI of 1 day. Page 7 of 48 O O OH OH OH OH O H C H 3 NH NH O H O NH 2 [

HCl

H2O] TABLE 2.1 Summary of Directions for Use of Kasugamycin.

Trade Name Formulation [ EPA Reg. Number] Application Type/ Timing and Equipment Application Rate ( lb ai/ A) Maximum Number of Applications per Season Maximum Seasonal Application Rate ( lb ai/ A) PHI ( Days) Use Directions and Limitations Proposed Use on Fruiting Vegetables ( Group 8) Imported from Mexico Kasumin ® 2L [ None] Foliar broadcast/ none specified 0.018 3 0.054 1 None specified 2.2 Structure and Nomenclature TABLE 2.2 Test Compound Nomenclature.

Chemical Structure Empirical Formula C14H25N3O9 [ C14H25N3O9° HCl° H2O]

Common Name Kasugamycin [ Kasugamycin hydrochloride hydrate]

Company Experimental Name TM 416 IUPAC Name ( Kasugamycin) 1L 1,3,4/ 2,5,6 1 deoxy 2,3,4,5,6 pentahydroxycyclohexyl 2 amino 2,3,4,6 tetradeoxy 4(   iminoglycino)   D arabino hexopyranoside or [ 5 amino 2 methyl 6( 2,3,4,5,6 pentahydroxycyclohexyloxy) tetrahydropyran 3 yl] amino   iminoacetic acid CAS Name ( Kasugamycin) 3 O[ 2 amino 4[( carboxyiminomethyl) amino] 2,3,4,6 tetradeoxy   D arabinohexopyranosyl D chiro inositol CAS Registry Number 6980 18 3 [ 19408 46 9]

End Use Product ( EUP) Kasumin ® 2L Chemical Class Aminoglycoside antibiotic fungicide Known Impurities of Concern None Page 8 of 48 2.3 Physical and Chemical Properties TABLE 2.3 Physicochemical Properties of the Technical Grade Compound ( Kasugamycin Hydrochloride Hydrate).

Parameter Value Reference Molecular Weight 433.8 PP# 3E6579 administrative materials ( MRIDs # 45910004 and  05) Melting Point/ Range 202 230  C ( decomposing)

pH 4.35 at 24.5  C ( 1% wt/ vol solution)

Density 0.43 g/ mL at 24.5  C Water Solubility g/ 100 mL pH 5 20.7 pH 7 22.8 pH 9 43.8 Solvent Solubility g/ 100 mL Methanol 0.744 Hexane < 1 x 10 5 Acetonitrile < 1 x 10 5 Methylene chloride < 1 x 10 5 Vapor Pressure < 0.013 mPa at 25  C Dissociation Constant ( pKa) pKa1 = 3.23 pKa2 = 7.73 pKa3 = 11.0 Octanol/ Water Partition Coefficient ( Log [ KOW]) < 1.96 at 23  C and pH 5 UV/ Visible Absorption Spectrum Not available 3.0 Metabolism Assessment 3.1 Comparative Metabolic Profile In the rat metabolism study, the mean radioactivity recovery 168 hours after exposure ranged from 91 to 97%, with the majority of the dose recovered within 48 hours in the feces ( 81.9 93.9%) and urine ( 1.26 3.07%). The maximum concentration found in the plasma of both males and females occurred approximately one hour after the administration of a single low or high dose. Between one and six hours after a single low or high dose, more kasugamycin accumulated in the kidneys, urinary bladder, and lymph nodes than in the blood, but after 168 hours, little or no kasugamycin was found in these tissues. The absorption and metabolism of kasugamycin in rats was limited ( less than 5% of the dose) and was not affected by sex, dose level, or duration of dosing. The parent compound was the major component identified in the urine, feces, liver, kidney, and plasma. Minor amounts ( less than 1% of the dose) of the Page 9 of 48 O O OH OH OH OH O H C H 3 NH NH O H O NH 2 Cl H O H 2 . .

* * * * *

* metabolite kasuganobiosamine were identified in urine, liver, kidney, and plasma, but none was detected in the feces. Elimination occurred primarily in the feces ( 88 to 95%), suggesting low absorption; kasugamycin was not excreted in the bile ( enterohepatic circulation did not occur). In the tomato metabolism study, the metabolite profile was similar for tomato fruit and foliage. Based on the results of the tomato metabolism study the petitioner proposes that the major metabolic pathway of kasugamycin in plants involves conjugation of the parent compound, conversion to kasugamycinic acid, and subsequent conjugation of kasugamycinic acid. Conversion of kasugamycin to 2 N acetyl kasugamycin and kasuganobiosamine was thought to be a minor metabolic route. Parent compound ( kasugamycin per se) was the major identified component in all samples from all harvest intervals.

3.2 Nature of the Residue in Foods 3.2.1. Description of Primary Crop Metabolism Arvesta Corporation has submitted a study investigating the metabolism of [ 14C] kasugamycin in tomatoes. The study is adequate for delineating the major residues. Based on the results of the tomato metabolism study the petitioner proposes that the major metabolic pathway of kasugamycin in tomatoes involves conjugation of the parent compound, conversion to kasugamycinic acid, and subsequent conjugation of kasugamycinic acid. Conversion of kasugamycin to 2 N acetyl kasugamycin and kasuganobiosamine was thought to be a minor metabolic route.

TABLE 3.2.1 Characteristics of Test Material Used in the Metabolism Study.

Chemical Structure Common Name [ 14C] Kasugamycin hydrochloride hydrate Radiolabel Position ( * ) All carbons, including the methyl substituent, in the 2,3,4,6 tetradeoxy   D arabino hexopyranosyl ring 3.2.2 Description of Livestock Metabolism There are no significant livestock feed items associated with this petition; therefore, the nature of the residue in livestock does not need to be addressed.

3.2.3 Description of Rotational Crop Metabolism Per the NAFTA Guidance Document on Data Requirements for Tolerances on Imported Commodities ( dated April 2003), rotational crop data are not required to support the proposed Page 10 of 48 O O OH OH OH OH O H C H 3 NH NH O H O NH 2 O O OH OH OH OH O H C H 3 NH O O H O NH 2 tolerances on imported fruiting vegetables.

3.3 Environmental Degradation Since kasugamycin is proposed for use only on imported fruiting vegetable commodities, with neither existing nor proposed US registration, there is no expectation that kasugamycin residues would occur in surface or ground water.

3.4 Tabular Summary of Metabolites and Degradates TABLE 3.4 Tabular Summary of Kasugamycin Metabolites and Degradates.

Common Name [ Code] % Total Radioactive Residues Chemical Name Chemical Structure Kasugamycin, Parent Compound [ TM 416]

Rat Essentially 100% TRR Tomato ( Fruit) 90 94% TRR ( 2 hour, 1 day) 69% TRR ( 7 day) 55 60% TRR ( 14, 21, 28 day) Tomato ( Foliage) 75 84% TRR ( 2 hour, 1 day) 70% TRR ( 7 day) 52 57% TRR ( 14, 21, 28 day) [ 5 amino 2 methyl 6 ( 2,3,4,5,6 pentahydroxycyclohexyloxy) tetrahydro pyran 3 yl] amino   iminoacetic acid Kasugamycinic acid [ KA 2]

Tomato ( Fruit) 10% TRR ( 21 day) 12% TRR ( 28 day) Tomato ( Foliage) 2 7% TRR ( all PHIs) N[ 5 amino 2 methyl 6 ( 2,3,4,5,6 pentahydroxycyclohexyloxy tetrahydropyran 3 yl] oxalamic acid TABLE 3.4 Tabular Summary of Kasugamycin Metabolites and Degradates.

Common Name [ Code] % Total Radioactive Residues Chemical Name Chemical Structure Page 11 of 48 O O OH OH OH OH O H C H 3 N H 2 NH 2 . 2 HCl O O OH OH OH OH O H C H 3 NH NH O H O NHCOCH 3 Kasuganobiosamine ° 2HCl [ KB 2]

Tomato ( Foliage) 1% TRR ( 1 to 28 day) 6( 3,5 diamino 6 methyltetrahydro pyran 2 yloxy) cyclohexane 1,2,3,4,5 pentol hydrochlorate 2 N acetyl kasugamycin [ KN 2]

Tomato ( Foliage) 1 2% TRR ( 14, 21, 28 day) [ 5 acetylamino 2 methyl 6 ( 2,3,4,5,6 pentahydroxycyclohexyloxy tetrahydropyran 3 ylamino] iminoacetic acid Tomato metabolism: MRID # 45910006. Single, foliar broadcast application of [ 14C] kasugamycin to tomato plants at 0.17 lb ai/ A ( roughly 3X the proposed maximum seasonal rate); fruit and foliage harvested at PHIs of 2 hours, 1, 7, 14, 21, and 28 days. Rat metabolism: MRID # 46030306. Oral gavage dosing of [ 14C] kasugamycin at a single low dose ( 100 mg/ kg bw); a single radiolabeled high dose ( 1000 mg/ kg bw); repeated unlabeled low doses ( 100 mg/ kg bw) for 14 days in the diet, plus 100 mg/ kg bw radiolabeled single dose; or repeated unlabeled high doses ( 1000 mg/ kg bw) for 14 days in the diet, plus 1000 mg/ kg bw radiolabeled single dose.

3.5 Toxicity Profile of Major Metabolites and Degradates No toxicity data were provided for the metabolites of kasugamycin that were observed in tomatoes.

3.6 Summary of Residues for Tolerance Expression and Risk Assessment TABLE 3.6 Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression.

Crop [ Matrix] Residue in Risk Assessment Residue in Tolerance Expression Fruiting Vegetables ( Crop Group 8) [ Fruit, Puree, Paste] Parent compound only, kasugamycin per se. Parent compound only, kasugamycin per se.

The only metabolite which would possibly be considered for inclusion in the ROC ( in addition to the parent) was kasugamycinic acid, which was present at less than 10% in all foliage Page 12 of 48 samples, and only occurred at concentrations of 10% or more in fruit samples from the 21 and 28 day PHIs. Because the petitioner is proposing a PHI of 1 day on imported fruiting vegetables, kasugamycinic acid is unlikely to occur as other than a minor constituent in imported fruiting vegetable commodities, and was therefore not included in the ROC for either tolerance expression or risk assessment purposes. Also, as residues of kasugamycin in all samples from the crop field trials ( at 1X) were either non detectable or less than the LOQ, the increase in risk from inclusion of this relatively minor metabolite was expected to be negligible.

4.0 Hazard Characterization/ Assessment 4.1 Hazard Characterization Kasugamycin is a fungicide that belongs to the aminoglycoside class of compounds, a group of antibiotics that inhibit protein synthesis. Unlike other members of this class, kasugamycin exhibits activity only towards phytopathogens and is ineffective against human and animal pathogens. As such, it is not used as a human or veterinary antibiotic. In general, the aminoglycosides are linked to nephrotoxicity. However, the only evidence of nephrotoxicity seen in the kasugamycin toxicology database was an increase in kidney lesions seen in females in a subchronic mouse study. An acute oral study was conducted using technical grade kasugamycin hydrochloride hydrate. Kasumin Liquid ( 2.02% ai), an end use product ( EUP), was tested for acute oral toxicity, dermal toxicity, eye irritation, dermal irritation, and skin sensitization. A second EUP, Kasumin 2 L ( 2.15% ai), was tested for acute inhalation toxicity. The technical material has low acute toxicity in rats ( Category IV for acute oral exposure). The EUPs also have low acute toxicity ( Category III for acute dermal exposure and Category IV for acute oral and inhalation exposures). The EUP tested is not a skin or eye irritant ( Category IV), nor is it a dermal sensitizer. Decreased body weights and body weight gains were observed following exposure to kasugamycin hydrochloride hydrate in multiple studies in the rat, mouse, dog, and rabbit. In the rat, body weight changes were reported in the subchronic, chronic, developmental, and twogeneration reproduction studies; effects were seen at lower doses in the longer term rat studies. In the mouse and the dog, decreases in body weight and body weight gain were seen in the subchronic studies at doses higher than the LOAEL; no treatment related effects, including body weight changes, were noted in either the chronic mouse or dog studies. The most sensitive species in terms of body weight changes was the rabbit. In the developmental rabbit study, the dose producing decreased body weights was at least an order of magnitude lower than the doses resulting in these changes in the studies utilizing other species. In the subchronic dog study, the decreases in body weights and body weight gains were most likely associated with treatment related effects on the tongue and mouth. These changes were seen in both sexes, were dose dependent, and included excessive salivation, swollen mouth with thickened skin, tongue erosions and ulcers, and histopathological lesions of the tongue. These effects may have contributed to the decrease in food consumption observed in this study, which likely led to the decrease in body weights and body weight gains. None of the mouth and tongue changes listed above were seen in the chronic dog study, although the highest dose tested Page 13 of 48 in the chronic study did cause those effects in the subchronic dog study. In the developmental rabbit study, the decreases in body weights and body weight gains were associated with decreased food consumption. In the main rabbit study, abortions occurred in animals following severe changes in body weights and food consumption. At the doses tested in this study, the abortions were not considered treatment related because the percentage of animals that aborted fell within the range of the historical control data. In two preliminary rabbit studies, in which higher doses were tested, there was a dose dependent increase in mortality and abortions that accompanied the effects on body weights. No increased quantitative or qualitative susceptibility was observed in the developmental rat or rabbit studies. In the developmental rat study, no developmental toxicity was observed at the highest dose tested, although this dose level induced toxicity in the dams including loose stool, distention of the large intestine, and decreased maternal body weights, body weight gains, and food consumption. In the main developmental rabbit study, neither offspring nor maternal toxicity was observed up to the highest dose tested. Although abortions were seen in the main study, the percentage of animals that aborted fell within the range of historical controls. Higher dose levels were tested in the preliminary developmental rabbit studies. In these studies, dose dependent increases in late term abortions ( and maternal mortality) were observed in animals following severe changes in maternal body weights associated with decreased food consumption. Maternal toxicity, indicated by the decreases in food consumption and body weights, was seen following multiple doses of kasugamycin hydrochloride hydrate. In the first preliminary rabbit developmental toxicity study, in which all treated animals were sacrificed in extremis, all of the animals survived for at least 8 days of dosing up to the limit dose ( 1000 mg/ kg/ day); there were no abortions in this study. Because the abortions only occurred after maternal toxicity developed following multiple doses of kasugamycin hydrochloride hydrate, the abortions seen in the rabbit developmental toxicity study are not attributable to a single oral dose. Increased qualitative or quantitative susceptibility was also not observed in the twogeneration reproduction study in rats. In this study, decreased parental generation male body weights and body weight gains were noted at a lower dose than the lowest dose at which effects on reproduction were observed. Reproductive toxicity was only observed in the F1 generation and included decreased fertility and fecundity in males and females, as well as an increased precoital interval during the mating period for the F2 litter. No offspring toxicity was found at any of the doses tested in the two generation reproduction study; however, offspring sexual maturation, organ weights, and histopathology were not examined. Testicular effects were noted in multiple studies. In the combined chronic/ carcinogenicity study in rats, the basis for the LOAEL was an increase in the incidence and severity of testicular tubular atrophy noted during the histopathological examinations at the end of the dosing period ( ie, week 104), as well as at two interim time points ( ie, weeks 26 and 52). Also in the chronic rat study, the incidences of testicular atrophy and softening observed grossly in males at the LOAEL were increased, compared to controls. As mentioned previously, reproductive toxicity ( ie, decreased fertility, decreased fecundity, and an increase in the pre coital interval during the mating period for the F2 litter) was seen in rats in the two generation reproduction study. In the reproduction study, small, fluid filled testes were observed in the F1 male rats. At the microscopic level, these animals had severe testicular atrophy and degeneration, with a complete loss of the germinal epithelium. In the subchronic mouse study, an increased number of high dose males had testicular tubular dilatation and degeneration compared to controls. No treatmentrelated effects, including testicular changes, were observed in the chronic mouse study; however, Page 14 of 48 the highest dose tested in this study was half of the dose selected as the LOAEL in the subchronic mouse study. In the subchronic dog study, 1/ 4 of the high dose dogs had hypospermia compared to 0/ 4 of the other dose groups. In the chronic dog study, 2/ 4 of the high dose males had testicular degeneration and 3/ 4 had chronic inflammation of the testes; only 1/ 4 of the controls had degeneration and 0/ 4 had inflammation. No mutagenic potential was noted in the following acceptable/ guideline mutagenicity studies: ( 1) the in vitro mammalian cell forward gene mutation test, ( 2) the mammalian erythrocyte micronucleus test, and ( 3) the unscheduled DNA synthesis in mammalian cells in culture. Additionally, no mutagenic potential was observed in an acceptable/ non guideline host mediated gene mutation assay. Two of the submitted mutagenicity studies were classified unacceptable. The bacterial gene mutation assay ( MRID # 45910028) was classified as such because kasugamycin hydrochloride hydrate was not tested up to the limit dose ( 5000

g/ plate); however, no mutagenic potential was noted up to the highest dose that was tested in the study ( 500

g/ plate). The structural chromosomal aberration study ( MRID # 45910025) was classified unacceptable because there were only 10 hours ( ie, less than 1 cell cycle) between the time from treatment to cell harvest, rather than the recommended 1.5 cell cycles ( ie, 16 to 21 hours); however, there was no evidence that kasugamycin hydrochloride hydrate induced a clastogenic effect. Although these two unacceptable mutagenicity studies create a data gap, in the absence of a carcinogenic effect in the rat and mouse studies and of a mutagenic effect in the available studies, the concern for mutagenesis is reduced. Therefore, on 4/ 13/ 2005, the Risk Assessment Review Committee ( RARC) recommended that the requirement for these mutagenicity studies be waived for the establishment of tolerances for kasugamycin on import commodities only. At this time, an acceptable bacterial gene mutation assay and an acceptable in vitro structural chromosomal aberration study will not further inform the risk assessment. No oncogenic potential was noted in the mouse oncogenicity study or in the rat combined chronic/ carcinogenicity study; additionally, no mutagenic potential was noted in any of the available mutagenicity studies. Based on the overall weight of the evidence, kasugamycin is classified as " not likely to be carcinogenic to humans." Other effects seen in the toxicology database for kasugamycin include anal lesions and perianal/ perigenital staining in the subchronic mouse study. Similarly, red and swollen skin around the anal opening and inflammation and ulceration of the rectum were noted in rats of both sexes and both generations in the two generation reproduction study. These effects may be related to the acidity of the active ingredient, which is primarily eliminated in the feces. The parent compound does not undergo significant metabolism; less than 1% of the administered dose was identified in the urine, liver, kidneys, and plasma as the metabolite kasuganobiosamine.

TABLE 4.1a Acute Toxicity Profile for Kasugamycin.

Test Material* [% ai] Guideline Number Study Type MRID Number Results Toxicity Category Technical Product [ 71] 870.1100 Acute oral  rat 45910012 LD50 (

+  ) > 5000 mg/ kg IV EUP [ 2.0] 870.1100 Acute oral  rat 45910014 LD50 (

+  ) > 5000 mg/ kg IV EUP [ 2.0] 870.1100 Acute oral  mouse 45910013 LD50 (

+  ) > 5000 mg/ kg IV TABLE 4.1a Acute Toxicity Profile for Kasugamycin.

Test Material* [% ai] Guideline Number Study Type MRID Number Results Toxicity Category Page 15 of 48 EUP [ 2.0] 870.1200 Acute dermal  rat 46030301 LD50 (

+  ) > 2000 mg/ kg III EUP [ 2.2] 870.1300 Acute inhalation  rat 45910018 LC50 (

+  ) > 4.892 mg/ L IV EUP [ 2.0] 870.2400 Acute eye irritation  rabbit 45910015 Mild eye irritant ( iritis at 1 hour, resolving by 24 hours; conjunctivitis at 1 hour, resolving by 24 hours). IV EUP [ 2.0] 870.2500 Acute dermal irritation  rabbit 45910017 Not irritating to the skin. IV EUP [ 2.0] 870.2600 Skin sensitization  guinea pig 45910016 Not a sensitizer under the conditions of this study. Not applicable * EUPs are formulated using kasugamycin hydrochloride hydrate; technical product ( Lot # KP 913) is 81% kasugamycin hydrochloride hydrate. Bracketed values are % ai as kasugamycin free base.

TABLE 4.1b Subchronic, Chronic, and Other Toxicity Profile for Kasugamycin.

Guideline Number Study Type/ Classification MRID Number Doses Results 870.3100 90 Day oral toxicity rodents  rat Acceptable/ guideline 45910020 0, 300, 1000, 3000, 6000 ppm M: 0, 17.5, 58.2, 176.7, 354.8 mg/ kg/ day F: 0, 20.3, 69.2, 201.0, 395.5 mg/ kg/ day NOAEL = 176.7/ 201.0 mg/ kg/ day ( M/ F) LOAEL = 354.8/ 395.5 mg/ kg/ day ( M/ F) based on decreased body weights and body weight gains.

870.3100 90 Day oral toxicity rodents  mouse Acceptable/ guideline 45910019 0, 300, 1000, 3000, 10000 ppm M: 0, 41.2, 135.4, 408.5, 1559 mg/ kg/ day F: 0, 58.0, 170.9, 565.6, 1834 mg/ kg/ day NOAEL = 135.4/ 170.9 mg/ kg/ day ( M/ F) LOAEL = 408.5/ 565.6 mg/ kg/ day ( M/ F) based on increased mortality and anal lesions ( M& F), and kidney lesions ( F).

At 1559/ 1834 mg/ kg/ day ( M/ F), decreased body weights and body weight gains ( M& F), testicular tubular dilatation and degeneration, perianal/ perigenital staining ( F), and extramedullary hematopoiesis of the spleen ( M) were seen. TABLE 4.1b Subchronic, Chronic, and Other Toxicity Profile for Kasugamycin.

Guideline Number Study Type/ Classification MRID Number Doses Results Page 16 of 48 870.3150 90 Day oral toxicity in nonrodents  dog Acceptable/ guideline 46030302 0, 300, 3000, 6000/ 0/ 4500* ppm M: 0, 10.6, 106.0, 182 mg/ kg/ day F: 0, 11.4, 107.9, 179 mg/ kg/ day * The high dose group was exposed to 6000 ppm on weeks 1 5, control diet on weeks 6 8, and 4500 ppm on weeks 8 13. NOAEL = 10.6/ 11.4 mg/ kg/ day ( M/ F) LOAEL = 106.0/ 107.9 mg/ kg/ day ( M/ F) based on tongue lesions, few feces, swollen mouth, excessive salivation, and thickened skin at the commissure of the mouth.

At 182/ 170 mg/ kg/ day ( M/ F), decreased body weights, body weight gains, and food consumption were seen.

870.3200 21/ 28 Day dermal toxicity Not applicable Not performed 870.3250 90 Day dermal toxicity Not applicable Not performed 870.3465 90 Day inhalation toxicity Not applicable Not performed 870.3700 Pre natal developmental in rodents  rat Acceptable/ guideline 45910022 0, 40, 200, 1000 mg/ kg/ day Maternal NOAEL = 200 mg/ kg/ day LOAEL = 1000 mg/ kg/ day based on decreased body weights, body weight gains, and food consumption; increased incidence of loose stool; and distention of the large intestine with stool in the cecum.

Developmental NOAEL = 1000 mg/ kg/ day LOAEL = > 1000 mg/ kg/ day 870.3700 Pre natal developmental in nonrodents  rabbit Acceptable/ guideline 46030303 0, 1, 3, 10 mg/ kg/ day Maternal NOAEL = 10 mg/ kg/ day LOAEL = > 10 mg/ kg/ day Note: Abortions and decreased maternal body weights, body weight gains, and food consumption were seen at 30 mg/ kg/ day in a range finding study.

Developmental NOAEL = 10 mg/ kg/ day LOAEL = > 10 mg/ kg/ day TABLE 4.1b Subchronic, Chronic, and Other Toxicity Profile for Kasugamycin.

Guideline Number Study Type/ Classification MRID Number Doses Results Page 17 of 48 870.3800 Reproduction and fertility effects  rat Acceptable/ guideline 45910023 0, 200, 1000, 6000 ppm M: 0, 13.7, 70.3, 425.3 mg/ kg/ day F: 0, 16.2, 82.9, 503.4 mg/ kg/ day Parental/ Systemic NOAEL = 13.7/ 16.2 mg/ kg/ day ( M/ F) LOAEL = 70.3/ 82.9 mg/ kg/ day ( M/ F) based on decreased body weights and body weight gains.

At 425.3/ 503.4 mg/ kg/ day ( M/ F), red and swollen skin around the anal opening ( M& F) and testicular atrophy/ degeneration in F1 males were seen.

Reproductive NOAEL = 70.3/ 82.9 mg/ kg/ day ( M/ F) LOAEL = 425.3/ 503.4 mg/ kg/ day ( M/ F) based on decreased fertility and fecundity in the F1 parents for both litters and increased pre coital interval during the mating period for the F2 litter.

Offspring NOAEL = 425.3/ 503.4 mg/ kg/ day ( M/ F) LOAEL = > 425.3/ 503.4 mg/ kg/ day ( M/ F)

870.4100 Chronic toxicity  rodents See 870.4300. This study includes requirements of both 870.4100 and 870.4200.

870.4100 Chronic toxicity  dog Acceptable/ guideline 46185901 0, 300, 1000, 3000 ppm M: 0, 10.5, 30.5, 99.6 mg/ kg/ day F: 0, 9.4, 33.4, 103.6 mg/ kg/ day NOAEL = 99.6/ 103.6 mg/ kg/ day ( M/ F) LOAEL = > 99.6/ 103.6 mg/ kg/ day ( M/ F)

870.4200 Carcinogenicity  rat See 870.4300. This study includes requirements of both 870.4100 and 870.4200.

870.4200 Carcinogenicity  mouse Acceptable/ guideline 46030304 0, 50, 300, 1500 ppm M: 0, 5.93, 34.94, 186.3 mg/ kg/ day F: 0, 7.25, 42.29, 215.2 mg/ kg/ day NOAEL = 186.3/ 215.2 mg/ kg/ day ( M/ F) LOAEL = > 186.3/ 215.2 mg/ kg/ day ( M/ F)

No evidence of carcinogenicity TABLE 4.1b Subchronic, Chronic, and Other Toxicity Profile for Kasugamycin.

Guideline Number Study Type/ Classification MRID Number Doses Results Page 18 of 48 870.4300 Combined chronic toxicity/ carcinogenicity  rat Acceptable/ guideline 45910024 0, 30, 300, 3000 ppm M: 0, 1.1, 11.3, 116 mg/ kg/ day F: 0, 1.4, 13.4, 140 mg/ kg/ day NOAEL = 11.3/ 140 mg/ kg/ day ( M/ F) LOAEL = 116/> 140 mg/ kg/ day ( M/ F) based on increased testicular softening and atrophy in males.

No evidence of carcinogenicity 870.5100 Gene mutation  bacterial reverse mutation assay Unacceptable/ upgradable 45910028 0, 5, 10, 50, 100, 500 ug/ plate for Salmonella typhimurium strain G46 ( his)

0, 5, 10, 50, 100, 200 ug/ plate for all other strains tested No mutagenic activity in bacteria ( Salmonella typhimurium and Escherichia coli) under conditions of this assay.

Not tested up to the limit dose, no indication of cytotoxicity, and no defined limit of solubility.

870.5300 Cytogenetics  in vitro mammalian cell gene mutation test ( CHO Cells)

Acceptable/ guideline 45910026 0, 0.5, 1, 2, 4, 6, 8, 10 mg/ ml No increase in mutant frequency at the HGPRT locus, in the presence or absence of S9 activation.

870.5375 Cytogenetics  in vitro mammalian cell chromosome aberration test Unacceptable/ not upgradable 45910025 0, 1, 2, 3, 4, 5 mg/ ml No increase in mutant frequency, in the presence or absence of S9 activation.

The time from treatment to cell harvest was insufficient.

870.5395 Cytogenetics  mammalian erythrocyte micronucleus test ( mice)

Acceptable/ guideline 46030305 0, 200, 1000, 5000 mg/ kg No evidence of induced chromosomal damage or other damage leading to micronucleus formation.

870.5550 Other effects  unscheduled DNA synthesis in mammalian cells in culture ( rats)

Acceptable/ guideline 45910027 First assay: 0 2.5 mg/ ml Second assay: 0 10 mg/ ml Third assay: 0 10 mg/ ml No evidence that unscheduled DNA synthesis was induced. TABLE 4.1b Subchronic, Chronic, and Other Toxicity Profile for Kasugamycin.

Guideline Number Study Type/ Classification MRID Number Doses Results Page 19 of 48 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ guideline 46030306 ( 1) 100 mg/ kg radiolabeled, single dose by oral gavage.

( 2) 100 mg/ kg unlabeled, 14 days in the diet, PLUS 100 mg/ kg radiolabeled, single dose by oral gavage.

( 3) 1000 mg/ kg radiolabeled, single dose by oral gavage.

( 4) 1000 mg/ kg unlabeled, 14 days in the diet, PLUS 1000 mg/ kg radiolabeled, single dose by oral gavage. The mean radioactivity recovery 168 hours after exposure ranged between 90.6 96.7%, with the majority of the dose recovered within 48 hours in the feces ( 81.9 93.9%) and urine ( 1.26 3.07%). The maximum concentration found in the plasma of both males and females occurred approximately one hour after the administration of a single low or high dose. Between one and six hours after a single low or high dose, more kasugamycin accumulated in the kidneys, urinary bladder, and lymph nodes than in the blood, but after 168 hours, little or no kasugamycin was found in these tissues. The absorption and metabolism of kasugamycin in rats was limited (< 5% dose) and was not affected by sex, dose level, or duration of dosing. Parent compound was the major component identified in the urine, feces, liver, kidney, and plasma. Minor amounts (< 1% dose) of the metabolite kasuganobiosamine were identified in urine, liver, kidney, and plasma, but none was detected in the feces. Elimination occurred primarily in the feces ( 87.7 94.5%); however, kasugamycin was not excreted in the bile ( enterohepatic circulation did not occur).

4.2 FQPA Hazard Considerations 4.2.1 Adequacy of the Toxicity Data Base The toxicology database for kasugamycin is considered adequate in terms of endpoint and dose response information to characterize any potential pre and/ or post natal risk for infants and children. Acceptable rat and rabbit developmental toxicity studies and an acceptable twogeneration reproduction study in the rat were submitted.

4.2.2 Evidence of Neurotoxicity There is no evidence that exposure to kasugamycin results in neurotoxicity. No clinical Page 20 of 48 signs of neurotoxicity, changes in brain weights, changes in brain histopathology, or any other neurotoxic effects were observed in any of the submitted studies, including the subchronic feeding studies, the chronic feeding studies, the developmental toxicity studies, or the two generation reproduction study.

4.2.3 Developmental Toxicity Studies No increased quantitative or qualitative susceptibility was observed in the developmental rat or rabbit studies. In the developmental rat study, no developmental toxicity was observed at the highest dose tested, although this dose level induced toxicity in the dams including loose stool, distention of the large intestine, and decreased maternal body weights, body weight gains, and food consumption. In the main developmental rabbit study, neither offspring nor maternal toxicity was observed up to the highest dose tested. Although abortions were seen in the main study, the percentage of animals that aborted fell within the range of historical controls. Higher dose levels were tested in the preliminary developmental rabbit studies. In these studies, dose dependent increases in late term abortions ( and maternal mortality) were observed in animals following severe changes in maternal body weights associated with decreased food consumption.

<IP_ADDRESS> Developmental Toxicity Study in Rats In a developmental toxicity study ( MRIDs # 45910022 and 45910021), kasugamycin hydrochloride hydrate ( 64.5% ai, Lot # KP 834) in purified water was administered daily by oral gavage to 24 female Crj: CD( SD) rats/ dose in a dose volume of 10 mL/ kg bw at dose levels of 0, 40, 200, or 1000 mg/ kg bw/ day on gestation days ( GD) 6 through 15. All dams were killed on GD 20; their fetuses were removed by cesarean section and examined. It was assumed, but not explicitly stated, that doses were adjusted for purity of the test compound. All animals survived to scheduled termination. For maternal toxicity, there were treatment related effects on clinical signs, body weight, and food consumption at 1000 mg/ kg/ day. An increased incidence ( p  0.01) of loose stool was sporadically observed during GD 8 to 15 in 7 high dose dams ( 9 observations), compared to none of the controls. An increased incidence ( p  0.05) of distention of the large intestine with stool in the cecum was observed in 5 of 24 high dose dams, compared with none of the controls. Body weights were decreased in the high dose group ( 3 to  7%; p  0.05) during GD 8 to 20, which resulted in decreased body weight gains during the dosing period ( 54% on GD 6 to 15; calculated by reviewers) and during the overall study ( 17% on GD 0 to 20; p  0.01). The gravid uterine weights of the high dose dams were non significantly decreased by 7%. When adjusted for gravid uterine weight, the body weights of these females were decreased on GD 20 ( 5%; p  0.05). Food consumption was decreased during the dosing period ( 24% on GD 6 to 15; p  0.05). The maternal LOAEL is 1000 mg/ kg/ day ( limit dose), based on decreased body weights, body weight gains, and food consumption, and increased incidences of loose stool and of distention of the large intestine with stool in the cecum. The maternal NOAEL is 200 mg/ kg/ day. For developmental toxicity, there were no abortions or premature deliveries. There were no treatment related effects on the number of litters, number of fetuses ( live or dead), number of resorptions ( early, late, or complete litter), post implantation loss, or fetal sex ratio. Although placental weights were decreased at 1000 mg/ kg/ day ( 12%; p  0.01), there were no treatmentrelated effects on fetal body weights, so this finding is considered equivocal. There were no Page 21 of 48 treatment related external, visceral, or skeletal malformations or variations. The developmental LOAEL was not observed. The developmental NOAEL is 1000 mg/ kg/ day ( limit dose). This study is classified acceptable/ guideline and satisfies the guideline requirements for a developmental toxicity study ( OPPTS 870.3700a; OECD 414) in the rat.

<IP_ADDRESS> Developmental Toxicity Study in Rabbits In a developmental toxicity study ( MRID # 46030303), kasugamycin hydrochloride hydrate ( 62.7% ai, Lot # KP 570) in distilled water was administered daily by oral gavage to 15 female New Zealand White rabbits/ dose in a dose volume of 10 mL/ kg at dose levels of 0, 1, 3, or 10 mg/ kg bw/ day on GD 6 through 19. All surviving does were killed on GD 29; their fetuses were removed by cesarean section and examined. In the main study, there were no treatment related effects on maternal mortality, clinical signs, body weight, food consumption, or gross pathology. Although effects were not seen at the highest dose tested, a new developmental toxicity study in the rabbit is not required because treatment related effects were seen at dose levels of 30 mg/ kg/ day and higher in the preliminary studies ( MRID # 46428701). The maternal LOAEL was not observed. The maternal NOAEL is 10 mg/ kg/ day. There were no treatment related effects on placental weight, fetal weight, sex ratio, postimplantation loss, or the numbers of litters, fetuses ( live or dead), or resorptions ( early, late, or complete litter). There were no treatment related external, visceral, or skeletal malformations, variations, or retardations. The developmental LOAEL was not observed. The developmental NOAEL is 10 mg/ kg/ day. This study is classified acceptable/ guideline and satisfies the guideline requirements for a developmental toxicity study ( OPPTS 870.3700b; OECD 414) in the rabbit.

4.2.4 Reproductive Toxicity Study In a two generation reproduction toxicity study ( MRID # 45910023), kasugamycin hydrochloride hydrate ( 81% ai; Lot # KP 913) was administered continuously in the diet to Crl: CD ® BR VAF/ Plus ® rats ( 25 rats/ sex/ dose) at dose levels of 0, 200, 1000, or 6000 ppm ( equivalent to 0/ 0, 13.7/ 16.2, 70.3/ 82.9, 425.3/ 503.4 mg/ kg bw/ day [ M/ F]). The P and F 1 parents were dosed for 10 weeks before they were mated to produce the F 1 and F 2a litters, respectively. The F 1 pups were weaned on post natal day ( PND) 21, and 25 pups/ sex/ group ( 1 pup/ sex/ litter as nearly as possible) were randomly selected as parents of the F 2 generation. Because of low fertility of the F 1 parents during the production of the F 2a litter, the F 1 adults were paired again six days following weaning of the F 2a litter to produce the F 2b litter. F 1 males that did not sire a litter in the first ( F 2a) mating were paired with proven females, and females that failed to deliver an F 2a litter were paired with proven males. In the P generation, 1000 ppm male body weights were decreased ( 6 to  7%; p  0.05) during Weeks 4 to 9 and Week 11, and 6000 ppm male body weights were decreased ( 5 to  6%; p  0.05) during Weeks 6 to 9. P generation cumulative body weight gains were decreased in the 1000 ppm males for Weeks 3 to 9 ( p  0.05) and in the 6000 ppm males for Weeks 3 to 14 ( p  0.05). In the F 1 generation, cumulative body weight gains were decreased in the 6000 ppm males ( p  0.05) during Weeks 1 to 3, 5 to 6, and 9 to 11 of the pre mating period for the F 2b litter. Red and swollen skin around the anal opening was observed exclusively in the 6000 ppm Page 22 of 48 group in both sexes and in both generations. This clinical sign was first noted at Week 10 in the P males ( 16 to 28%) and P females ( 13 to 100%), and it was first seen at Week 5 in the F 1 males ( 8 to 75%) and F 1 females ( 12 to 100%). Gross examination of the animals in this dose group revealed red foci/ areas on the rectum in both sexes and both generations ( 80 to 100% treated vs 0 controls), as well as thickened walls of the rectum in the F 1 males ( 46% treated vs 0 controls). Microscopically, chronic active inflammation and ulceration of the rectum were observed in both sexes of both generations ( 88 to 100% treated vs 0 controls). Additionally, in the P males at this dose, squamous cell hyperplasia was observed at the ano rectal junction ( 38% treated vs 0 controls). At 6000 ppm, testes that were small ( 38%) and containing fluid ( 67%) were observed ( compared to 0% of controls) in the F 1 males at necropsy. Microscopically, atrophy/ degeneration was noted in the testes in the P ( 12% treated vs 0% controls) and F 1 ( 63% treated vs 0% controls) males. This atrophy/ degeneration was marked to severe in the F 1 generation and was characterized by complete loss of the germinal epithelium, atrophy of the seminiferous tubules, persistence of Sertoli cells, and variable amounts of intertubular edema. Additionally in the 6000 ppm F 1 males, pelvic dilatation of the kidney and chronic progressive nephropathy were observed microscopically ( 21% treated vs 4% controls). The LOAEL for parental toxicity is 1000 ppm ( equivalent to 70.3/ 82.9 mg/ kg/ day [ M/ F]), based on decreased body weights and body weight gains in the P generation males. The NOAEL is 200 ppm ( equivalent to 13.7/ 16.2 mg/ kg/ day [ M/ F]). In F 1 female pups, adjusted body weights were decreased ( 6%; p  0.05) at 6000 ppm on PND 21. This decrease was minor and occurred only at the end of the lactation period. Furthermore, there were no treatment related effects on body weights in the F 2a or F 2b females or in the male pups of either generation at this dose. Thus, this decrease is not considered adverse. Pup weights at  1000 ppm in both sexes and generations were comparable to controls. It should be noted that several parameters were not examined in the offspring, including sexual maturation, developmental landmarks, organ weights, and histopathology. Based on the parameters investigated, the LOAEL for offspring toxicity was not observed. The NOAEL is 6000 ppm ( equivalent to 425.3/ 503.4 mg/ kg/ day). In the F 1 generation, male and female fertility and fecundity indices were decreased at 6000 ppm for the F 2a litter ( 64 to 67% treated vs 88 to 96% controls) and the F 2b litter ( 36 to 39% treated vs 88 to 100% controls). Additionally, in the F 1 generation, the pre coital interval during the mating period to produce the F 2b litter was longer ( p  0.01) at 6000 ppm ( 5.73 days) compared to controls ( 2.23 days). The LOAEL for reproductive toxicity was 6000 ppm ( equivalent to 425.3/ 503.4 mg/ kg/ day [ M/ F]) based on decreased fertility and fecundity in the F1 parents for both litters and increased pre coital interval during the mating period for the F2 litter. The NOAEL is 1000 ppm ( equivalent to 70.3/ 82.9 mg/ kg/ day [ M/ F]). This study is classified as acceptable/ guideline and satisfies the Guideline requirements ( OPPTS 870.3800; OECD 416) for a two generation reproduction study in the rat.

4.2.5 Additional Information from Literature Sources Studies available in the open literature include articles on the mechanism of resistance and toxicity of kasugamycin towards various species of bacteria.

4.2.6 Pre and/ or Post Natal Toxicity Page 23 of 48 <IP_ADDRESS> Determination of Susceptibility No increased quantitative or qualitative susceptibility was observed in the developmental rat or rabbit studies or in the two generation reproduction study. No offspring toxicity was observed at any of the doses tested in these three studies. Reproductive toxicity was noted in the F1 generation of the two generation reproduction study. However, because parental toxicity ( decreased body weights and body weight gains) occurred at a lower dose than that which resulted in effects on reproduction, there is no increased quantitative or qualitative susceptibility of the offspring.

<IP_ADDRESS> Degree of Concern Analysis and Residual Uncertainties for Pre and/ or Post Natal Susceptibility The toxicology database for kasugamycin is complete with respect to pre and post natal toxicity and shows no evidence of increased qualitative or quantitative susceptibility in the offspring. Therefore, there are no residual uncertainties for pre and/ or post natal toxicity.

4.3 Recommendation for a Developmental Neurotoxicity Study 4.3.1 Evidence that Supports Requiring a Developmental Neurotoxicity Study There is no evidence to support the requirement for a developmental neurotoxicity study on kasugamycin.

4.3.2 Evidence that Supports not Requiring a Developmental Neurotoxicity Study There was no evidence of neurotoxicity in any of the studies available in the toxicology database, including the subchronic feeding studies, the chronic feeding studies, the developmental toxicity studies, and the two generation reproduction study. Therefore, acute and subchronic neurotoxicity studies are not required. A developmental neurotoxicity study is not required because: ( 1) there are no data gaps for the assessment of the effects of kasugamycin following in utero and/ or post natal exposure, ( 2) there is no evidence of neurotoxicity in the available toxicology studies, and ( 3) there is no increased quantitative or qualitative susceptibility noted in either the rat or rabbit developmental toxicity studies or in the two generation reproduction study.

4.4 Hazard Identification and Toxicity Endpoint Selection 4.4.1 Acute Reference Dose ( aRfD)  Females ( 13 to 49 Years of Age) No appropriate dose and endpoint could be identified for acute dietary exposure in Females ( 13 49). No pre natal or fetal toxicity attributable to a single oral dose was observed in the developmental toxicity studies in rats or rabbits or in the two generation reproduction study in rats. In the main developmental rabbit study, offspring toxicity was not observed up to the highest dose tested. Although abortions were seen in the main study, the percentage of animals that aborted fell within the range of the historical control data. Higher dose levels were tested in the preliminary developmental rabbit studies. In these studies, dose dependent increases in late Page 24 of 48 term abortions ( and maternal mortality) were observed in animals following severe changes in maternal body weights associated with decreased food consumption. Maternal toxicity, indicated by the decreases in food consumption and body weights, was seen following multiple doses of kasugamycin hydrochloride hydrate. In the first preliminary rabbit developmental toxicity study, in which all treated animals were sacrificed in extremis, all of the animals survived for at least 8 days of dosing up to the limit dose ( 1000 mg/ kg/ day); there were no abortions in this study. Because the abortions only occurred after maternal toxicity developed following multiple doses of kasugamycin hydrochloride hydrate, the abortions seen in the rabbit developmental toxicity study are not attributable to a single oral dose.

4.4.2 Acute Reference Dose ( aRfD)  General Population No appropriate dose and endpoint could be identified for acute dietary exposure in the general population, including infants and children. No neurotoxic effects, maternal toxicity following 1 or 2 doses in the developmental studies, or other effects were seen following a single oral dose that could be relevant for this population group.

4.4.3 Chronic Reference Dose ( cRfD) Study Selected: Combined chronic toxicity/ carcinogenicity study in rats. MRID Number: 45910024. Executive Summary: In a combined chronic toxicity/ carcinogenicity study ( MRID # 45910024), kasugamycin hydrochloride hydrate ( 67.1% ai, dose levels adjusted for purity; Lot # KP 570) was administered to 70 Wistar rats/ sex/ dose in the diet at dose levels of 0, 30, 300, or 3000 ppm ( equivalent to 0/ 0, 1.1/ 1.4, 11.3/ 13.4, and 115.9/ 139.8 mg/ kg bw/ day in males/ females) for up to 2 years. Ten rats/ sex/ dose were sacrificed at Weeks 26 and 52, and the remaining survivors were sacrificed at Week 104. No treatment related effects were observed on mortality, clinical signs, food consumption or efficiency, water consumption, ophthalmologic examination, hematology, clinical chemistry, or urinalysis. Adverse findings were confined to the testes in the 3000 ppm males. Non statistically significant increased incidences of testicular softening and atrophy were observed grossly at interim sacrifice on Week 26 ( n= 10; 20 to 30% treated vs 0% controls), at interim sacrifice on Week 52 ( n= 10; 30% treated vs 0% controls), and when all males were combined on Week 104 ( n= 69 70; 19 to 20% treated vs 11% controls). Non statistically significant increased incidences of unilateral or bilateral testicular tubular atrophy were observed as follows: ( 1) slight to moderate at Week 26 ( 30% treated vs 0% controls), ( 2) severe at Week 52 ( 30% treated vs 0% controls), and ( 3) slight to severe when all males were combined ( n= 69 70; 25% treated vs 14% controls). Severity of the tubular atrophy in all males was also dependent on the dose. Additionally, in a concurrently submitted 2 generation reproduction study in rats ( MRID # 45910023), the testes were adversely affected in both the parents and offspring, with signs including testicular atrophy/ degeneration, small testes, and seminiferous tubule atrophy. Although gross and microscopic testicular atrophy is common in old rats, increased atrophy at the interim sacrifices suggests a treatment related effect. Body weights were slightly and transiently decreased in the 3000 ppm females, resulting in minor decreases in cumulative body weight gains during Weeks 0 to 13 and 0 to 52. Overall body Page 25 of 48 weight gains ( Weeks 0 to 104) were similar to controls. As the effects on body weight gains were slight and only occurred during the first year of the study, these findings were not considered adverse. Additionally at 3000 ppm, several findings were observed in the kidney, lungs, nose, and liver that were considered to be unrelated to treatment. Relative ( to body) kidney weights were increased slightly in both sexes at Week 104. Increased incidences of lipofuscin deposition of proximal tubular cells ( usually bilateral) of the kidneys were observed as follows: ( 1) in the males at Week 26, ( 2) in both sexes at Week 52, and ( 3) in both sexes when all animals were combined. There was no indication that the etiology of the lipofuscin deposition was related to treatment. Without supporting clinical chemistry, urinalysis, or gross/ microscopic pathology to corroborate nephrotoxicity, the slight increases in relative kidney weight were considered to be incidental. Increased incidences of foam cell aggregation in the lungs were observed in the males at Week 52, and in both sexes when all animals were combined. No further evidence of toxicity to the lung was observed during gross or microscopic pathology; therefore, these findings were considered to be incidental. An increased incidence of slight to moderate rhinitis vs slight in controls was observed when all treated males were combined. Considering the nature of the lesion, and slight increase in frequency and severity, this minor condition was not considered adverse. Total cholesterol was slightly decreased in the 3000 ppm females after 52, 78, and 104 weeks of treatment. However, this slight decrease may be within the range of natural variation ( historical control data were not provided). When all animals were combined, acidophilic cell foci of cellular alteration in the liver was observed in the females at 300 ppm or higher; however, the effect was not clearly related to dose and was considered equivocal. Additionally in the females, increased incidences of slight hepatocellular atrophy were observed at Week 52, and when all females were combined. Without any evidence of a clear hepatotoxic effect, the hepatocellular atrophy was considered to be incidental. The LOAEL is 116 mg/ kg/ day in males, based on increased testicular softening and atrophy; the LOAEL in females was not observed. The NOAEL is 11.3/ 140 mg/ kg/ day in males/ females. At the doses tested, there was not a treatment related increase in tumor incidence when compared to controls. Dosing was considered adequate based on increased lesions in the testes. This study is classified acceptable/ guideline and satisfies the guideline requirements for a combined chronic toxicity/ carcinogenicity study ( OPPTS 870.4300; OECD 453) in the rat. Dose and Endpoint for Establishing cRfD: 11.3 mg/ kg/ day ( NOAEL) based on increased testicular softening and atrophy in males at 116 mg/ kg/ day ( LOAEL). Uncertainty Factor( s): 100 ( 10X for interspecies extrapolation and 10X for intraspecies variations). Comments about Study/ Endpoint/ Uncertainty Factor: Although they did not reach statistical significance, the testicular effects observed in high dose male rats in the combined chronic/ carcinogenicity study were selected as the basis for the study LOAEL, as well as for the chronic dietary endpoint, because an increase in the incidence and severity of these effects were noted and because similar effects were seen across species in multiple studies. In the chronic rat study, the incidences of testicular tubular atrophy observed histopathologically at week 26, week 52, and when males at all time points were combined were 30%, 30%, and 25%, respectively, of Page 26 of 48 the high dose group and 0%, 0%, and 14%, respectively, of the controls. Additionally, there was an increase in severity of the histopathological testicular tubular atrophy. When all males were combined, 9% of the controls and 6% of the high dose group had slight testicular atrophy, while 6% of the controls and 19% of the high dose males had moderate or severe testicular atrophy. Testicular tubular atrophy and gross testicular changes were also observed in rats in the twogeneration reproduction study; see the next paragraph for more details. In the subchronic mouse study, 7/ 10 and 4/ 10 of the high dose males had tubular dilatation and degeneration, respectively, compared to 0/ 10 and 0/ 10 of the controls. Although these testicular effects were not replicated in the chronic mouse study, the highest dose tested in the chronic mouse study was half the value of the LOAEL for the subchronic mouse study. In the subchronic dog study, 1/ 4 of the high dose dogs had hypospermia compared to 0/ 4 of the other dose groups. In the chronic dog study, 2/ 4 of the high dose males had testicular degeneration and 3/ 4 had chronic inflammation; only 1/ 4 of the controls had degeneration and 0/ 4 had inflammation. The two generation reproduction study in rats ( MRID # 45910023) was considered a cocritical study. The executive summary for this study is found in Section 4.2.4. In the twogeneration reproduction study, the LOAEL for parental toxicity was 1000 ppm ( equivalent to 70.3/ 82.9 mg/ kg/ day [ M/ F]), based on decreased body weights and body weight gains seen in the P generation males. The NOAEL for this study is 200 ppm ( equivalent to 13.7/ 16.2 mg/ kg/ day [ M/ F]). Testicular effects similar to those found in the combined chronic/ carcinogenicity study in rats were also observed in this study at a dose greater than the reproduction study LOAEL. At 425 mg/ kg/ day, testicular tubular atrophy was seen in 12% of the parental males and in 63% of the F1 generation males, compared to none of the control P or F1 males. Grossly, at this same dose, 38% of the F1 generation had small testes. The F1 male fertility index decreased to 38% at 425 mg/ kg/ day compared to 88% in controls. The NOAEL and LOAEL from the subchronic dog study ( 10.6 and 106.0 mg/ kg/ day, respectively) are slightly lower than the NOAEL and LOAEL from the combined chronic/ carcinogenicity study in rats ( 11.3 and 116 mg/ kg/ day, respectively). However, this study was not selected to establish the cRfD because the effects used as a basis for the LOAEL in the subchronic dog study ( ie, excessive salivation, swollen mouth with thickened skin, tongue erosions and ulcers, histopathological lesions of the tongue, and few feces) were not seen at similar doses tested in the chronic dog study. Although the treatment related effects in the subchronic study were not used to establish the cRfD, the selected NOAEL is protective of these effects.

Chronic RfD = 11.3 mg/ kg/ day ( NOAEL) = 0.113 mg/ kg/ day 100 ( UF)

4.4.4 Incidental Oral Exposure ( Short and Intermediate Term) Because no proposed uses would result in residential exposure in the US, no incidental oral exposure assessment is required at this time.

4.4.5 Dermal Absorption Because no proposed uses would result in residential or occupational exposure in the US, no dermal exposure assessment is required at this time. As such, dermal absorption and dermal toxicity studies are currently not required, and a dermal absorption factor is not currently needed. Page 27 of 48 4.4.6 Dermal Exposure ( Short, Intermediate and Long Term) Because no proposed uses would result in residential or occupational exposure in the US, no dermal exposure assessment is required at this time.

4.4.7 Inhalation Exposure ( Short, Intermediate and Long Term) Because no proposed uses would result in residential or occupational exposure in the US, no inhalation exposure assessment is required at this time.

4.4.8 Margins of Exposure Because no proposed uses would result in residential or occupational exposure in the US, no incidental oral, dermal, or inhalation exposure assessments are required at this time. Therefore, it is not currently necessary to set levels of concern for margins of exposure.

4.4.9 Recommendation for Aggregate Exposure Risk Assessments Because no proposed uses would result in residential exposure in the US, an aggregate exposure assessment including any exposures other than dietary ( food only) is not required at this time. Based on the proposed tolerances on imported commodities, exposure to kasugamycin will not occur via the drinking water, incidental oral, dermal, or inhalation pathways.

4.4.10 Classification of Carcinogenic Potential The risk assessment team has classified kasugamycin as " not likely to be carcinogenic to humans." No oncogenic potential was noted in the mouse oncogenicity or in the rat combined chronic/ carcinogenicity studies; additionally, no mutagenic potential was noted in any of the five mutagenicity studies.

TABLE 4.4 Summary of Toxicological Doses and Endpoints for Kasugamycin to be Used in Human Health Risk Assessments.

Exposure Scenario Dose Used in Risk Assessment and UF 1 Special FQPA SF 2 and Level of Concern for Risk Assessment Study and Toxicological Effects Acute Dietary ( females 13 to 49 years of age) None None Not selected No appropriate dose and endpoint could be identified for these population groups.

Acute Dietary ( general population including infants and children) None None Not selected No appropriate dose and endpoint could be identified for these population groups.

Chronic Dietary ( all populations) NOAEL = 11.3 mg/ kg/ day UF = 100 Chronic RfD = 0.113 mg/ kg/ day FQPA SF = 1 cPAD = chronic RfD FQPA SF = 0.113 mg/ kg/ day Combined chronic toxicity/ oncogenicity study in rats LOAEL = 116 mg/ kg/ day based on increased testicular softening and atrophy. TABLE 4.4 Summary of Toxicological Doses and Endpoints for Kasugamycin to be Used in Human Health Risk Assessments.

Exposure Scenario Dose Used in Risk Assessment and UF 1 Special FQPA SF 2 and Level of Concern for Risk Assessment Study and Toxicological Effects Page 28 of 48 Incidental Oral ( all durations) None None Not selected Tolerance on imported commodities  no proposed uses would result in residential exposure in the US.

Dermal ( all durations) None None Not selected Tolerance on imported commodities  no proposed uses would result in residential or occupational exposure in the US.

Inhalation ( all durations) None None Not selected Tolerance on imported commodities  no proposed uses would result in residential or occupational exposure in the US.

Cancer ( oral, dermal, inhalation) Classification: No oncogenic potential was noted in the mouse oncogenicity or in the rat combined chronic/ carcinogenicity studies; additionally, no mutagenic potential was noted in any of the five mutagenicity studies. Classification of kasugamycin is " not likely to be carcinogenic to humans".

1. UF = Uncertainty Factor. 2. FQPA SF = special FQPA Safety Factor ( refer to Section 4.5). 3. NOAEL = No Observed Adverse Effect Level. 4. RfD = Reference Dose. 5. PAD = Population Adjusted Dose ( a = acute, c = chronic). 6. LOAEL = Lowest Observed Adverse Effect Level.

4.5 Special FQPA Safety Factor Based on the hazard and exposure data, the kasugamycin risk assessment team has recommended that the special FQPA SF be reduced to 1X because there are no/ low concerns and no residual uncertainties with regard to pre and/ or post natal toxicity. This recommendation is based on the following: ( 1) there are no data gaps for the assessment of the effects of kasugamycin following in utero and/ or post natal exposure; a developmental neurotoxicity study is not required; ( 2) there is no indication of increased quantitative or qualitative susceptibility of rats or rabbits to in utero and/ or post natal exposure to kasugamycin; ( 3) the acute and chronic dietary food exposure assessments utilize proposed tolerance level or higher residues and 100% crop treated information for all commodities; by using these screening level assessments, acute and chronic exposures/ risks will not be underestimated; and ( 4) there are no existing or proposed residential uses for kasugamycin at this time.

4.6 Endocrine Disruption EPA is required under the FFDCA, as amended by FQPA, to develop a screening program Page 29 of 48 to determine whether certain substances ( including all pesticide active and other ingredients) " may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effects as the Administrator may designate." Following recommendations of its Endocrine Disruptor and Testing Advisory Committee ( EDSTAC), EPA determined that there was a scientific basis for including, as part of the program, the androgen and thyroid hormone systems, in addition to the estrogen hormone system. EPA also adopted EDSTAC's recommendation that the Program include evaluations of potential effects in wildlife. For pesticide chemicals, EPA will use FIFRA and, to the extent that effects in wildlife may help determine whether a substance may have an effect in humans, FFDCA authority to require the wildlife evaluations. As the science develops and resources allow, screening of additional hormone systems may be added to the Endocrine Disruptor Screening Program ( EDSP). For kasugamycin, effects which indicate potential endocrine disruption include changes in the testes ( eg, lesions, softening, atrophy, and degeneration) in rats and mice. Decreased fertility, decreased fecundity, and an increased pre coital interval during the mating period were seen in rats; these effects may be reflections of the changes in the testes. When additional appropriate screening and/ or testing protocols being considered under the Agency's EDSP have been developed, kasugamycin may be subjected to further screening and/ or testing to better characterize effects related to endocrine disruption.

5.0 Public Health Data Kasugamycin is a new active ingredient and, as such, no public health data are currently available. Kasugamycin operates via a mode of action different from that of the other aminoglycoside antibiotics such as streptomycin. Because kasugamycin is active only against phytopathogenic fungi and bacteria, it has never been employed as a human or veterinary use antibiotic. HED is aware that FDA and CDC have concerns regarding the potential for antibiotics to induce bacterial resistance arising from their use as pesticides. HED has met with these agencies recently to discuss resistance issues and an ongoing dialogue is anticipated. HED's level of concern is low regarding development of resistance ( associated with kasugamycin's use as a fungicide) arising from tolerances for kasugamycin on imported fruiting vegetables because: ( 1) proposed use rates for kasugamycin are low, and residues following its application are either very low or non detectable, ( 2) the proposed uses are only on imported fruiting vegetables, with no proposed domestic uses, and ( 3) there are no human or veterinary uses of kasugamycin as an antibiotic.

6.0 Exposure Characterization/ Assessment 6.1 Dietary Exposure/ Risk Pathway 6.1.1 Residue Profile The submitted crop field trial data for peppers and tomatoes are adequate to support the proposed uses on fruiting vegetables. Residues of kasugamycin were below the method LOQ ( less than 0.040 ppm) in/ on all samples of peppers and tomatoes harvested 1 day following the Page 30 of 48 last of three foliar broadcast applications of the Kasumin ® 2L liquid formulation at total seasonal rates of 0.052 to 0.054 lb ai/ A. In residue decline trials, residues were non detectable in/ on all samples of peppers and tomatoes collected 0, 1, 3, and 7 days after final application; therefore, it is not possible to make a conclusion concerning whether kasugamycin residues decline in fruiting vegetables with increasing sampling intervals following treatment. The tomato field trials included both processing and fresh market tomato varieties; however, no small varieties of tomatoes ( cherry tomatoes) were included. The studies are supported by adequate storage stability data. Processing studies on tomato, for the processed commodities tomato puree and tomato paste ( residue data on paste cover tomato juice), were not conducted; a request for waiver of the requirement for tomato processing data was submitted. Based on the results of field trial data at the 5X exaggerated rate, residue data on small tomato varieties and a tomato processing study are not required. The proposed residue analytical method is adequate for data collection; however, radiovalidation data were not submitted. The method was submitted to ACL/ BEAD for a PMV trial ( PMV Request Memo, D313672, William T. Drew, 3/ 1/ 2005), which it subsequently failed ( PMV Results Memo, D313673, Patricia G. Schermerhorn, 7/ 13/ 2005). The method includes instructions for the analysis of tomato and pepper samples. Briefly, macerated or ground frozen samples are extracted twice with methanol/ water ( 7: 3 vol/ vol) at pH 4. The extracts are filtered, combined, and concentrated, then filtered through diatomaceous earth to remove precipitates. The filtered concentrate is diluted with water and purified using two separate ion exchange columns. The resulting purified extract is concentrated before analysis via reverse phase, ionpairing HPLC/ UV analysis. The validated LOQ is 0.040 ppm, determined as the lower limit of method verification ( LLMV), and the calculated limit of detection ( LOD) is 0.013 ppm. The method does not include a confirmatory method, and no interference study was conducted.

6.1.2 Acute and Chronic Dietary Exposure and Risk The dietary exposure assessment, entitled Kasugamycin Chronic Dietary Exposure Assessment for the Import Tolerance on Fruiting Vegetables, was conducted by Douglas A. Dotson, 5/ 9/ 2005, DP Barcode D315531. A chronic dietary risk assessment was conducted for the new active ingredient kasugamycin using the Dietary Exposure Evaluation Model ( DEEM FCID, Version 2.03), which uses food consumption data from the USDA's Continuing Surveys of Food Intakes by Individuals ( CSFII) from 1994 to 1996 and 1998. The analysis was performed to support requests for tolerances on imported fruiting vegetables ( Crop Group 8). Acute Dietary Exposure: An acute dietary exposure analysis was not performed for kasugamycin, because no appropriate dose or endpoint could be identified for acute dietary exposure in the general population or any of the population subgroups. Chronic Dietary Exposure Results and Characterization: A conservative chronic dietary exposure analysis was performed for kasugamycin. The analysis is based on tolerance level residues ( modified by DEEM default processing factors) and the assumption that 100% of the crop will be treated. The risk estimates for all population subgroups are below HED's level of concern. The risk estimate for the general US population is less than 1% of the cPAD. The most highly exposed population subgroup is Children ( 1 2), which utilizes less than 1% of the cPAD. Page 31 of 48 TABLE 6.1 Summary of Dietary Exposure and Risk for Kasugamycin.

Population Subgroup Acute Dietary Chronic Dietary 1 aPAD ( mg/ kg) Exposure ( mg/ kg/ day) % aPAD cPAD ( mg/ kg/ day) Exposure ( mg/ kg/ day) 2 % cPAD General US Population Not applicable. 0.113 0.000073 < 1 All Infants < 1 year 0.113 0.000040 < 1 Children 1 2 years 0.113 0.00018 < 1 Children 3 5 years 0.113 0.00016 < 1 Children 6 12 years 0.113 0.00011 < 1 Youths 13 19 years 0.113 0.000075 < 1 Adults 20 49 years 0.113 0.000063 < 1 Adults 50+ years 0.113 0.000050 < 1 Females 13 49 years 0.113 0.000060 < 1 1. Values for the population with the highest risk for each type of risk assessment are bolded. 2. Reported to 2 significant figures.

6.2 Water Exposure/ Risk Pathway Since kasugamycin is proposed for use only on imported fruiting vegetable commodities, the sole anticipated exposure route for the US population is via dietary ( food) exposure. With no proposed US registration, there is no expectation that kasugamycin residues would occur in surface or ground water sources of drinking water.

6.3 Residential ( Non Occupational) Exposure/ Risk Pathway Since kasugamycin is proposed for use only on imported fruiting vegetable commodities, the sole anticipated exposure route for the US population is via dietary ( food only) exposure. With no proposed US registration, kasugamycin is not intended for use in public or residential settings. Therefore, residential exposure is not expected and no residential risk assessment was performed.

7.0 Aggregate Risk Assessments and Risk Characterization Since kasugamycin is proposed for use only on imported fruiting vegetable commodities, the sole anticipated exposure route for the US population is via dietary ( food only) exposure. With no proposed US registration, there is no expectation that exposure to kasugamycin residues would occur via water consumption or residential use. Therefore, aggregate exposure is not expected and no aggregate risk assessment was performed.

8.0 Cumulative Risk Characterization/ Assessment Unlike other pesticides for which EPA has followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to kasugamycin and any other substances, and kasugamycin does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, Page 32 of 48 EPA has not assumed that kasugamycin has a common mechanism of toxicity with other substances. For information regarding EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy statements released by EPA's Office of Pesticide Programs ( OPP) concerning common mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA's website at http:// www. epa. gov/ pesticides/ cumulative/.

9.0 Occupational Exposure/ Risk Pathway Since kasugamycin is proposed for use only on imported fruiting vegetable commodities, the sole anticipated exposure route for the US population is via dietary ( food) exposure. With no proposed US registration, there is no expectation that exposure to kasugamycin residues would occur via occupational use. Therefore, no occupational risk assessment was performed.

10.0 Data Needs and Label Requirements 10.1 Toxicology No additional toxicological studies are required for kasugamycin at this time.

10.2 Residue Chemistry HED has examined the residue chemistry database for kasugamycin and recommends against the establishment of tolerances for residues of this fungicide in/ on imported fruiting vegetables until the data deficiencies listed below ( OPPTS Residue Chemistry Test Guideline 860.1340) have been resolved. The fact that an adequate analytical method for tolerance enforcement is unavailable does not impact this risk assessment regarding risk characterization. 860.1340 Residue Analytical Method ( Plant Commodities) ( 1) The proposed enforcement method should be modified to include a confirmatory analysis method; alternatively, the petitioner may submit an interference study with all pesticides for which tolerances on tomatoes and/ or peppers have been established. ( 2) The method has failed a PMV trial ( PMV Results Memo, D313673, Patricia G. Schermerhorn, 7/ 13/ 2005), and should be modified to include the revisions recommended by ACL. 860.1550 Proposed Tolerances The available crop field trial data support a tolerance for residues of kasugamycin per se in/ on imported fruiting vegetable commodities ( Crop Group 8) at 0.04 ppm, the method's LOQ. Based on the submitted field trial data, the following use pattern would be supported for fruiting vegetables: application of the Kasumin ® 2L formulation as a foliar broadcast spray in a minimum spray volume of 30 GPA using ground equipment, with no spray adjuvants, a minimum RTI of 3 days, and a PHI of 1 day. HED recommends that the petitioner's request for waiver of tomato processing data be granted; a tomato processing study is not required. The exaggerated rate ( 5X) tomato field trial data indicate that separate tolerances for tomato juice, tomato puree and tomato paste are not required. The proposed tolerances should be revised to reflect the tolerance as recommended by HED and the correct commodity definition as specified in Table 10.2, below. The petitioner should submit a revised Section F for PP# 3E6579 to reflect these changes. Page 33 of 48 TABLE 10.2 Tolerance Summary for Kasugamycin.

Commodity Proposed Tolerance ( ppm) Recommended Tolerance ( ppm) Comments/ Correct Commodity Definition Fruiting Vegetables ( Crop Group 8) 0.04 0.04 Vegetable, fruiting, group 8 Tomato Juice 0.06 None Residues are not expected to exceed the tolerance on the RAC. Tomato Puree 0.06 Tomato Paste 0.25 References: 1. Kasugamycin Chronic Dietary Exposure Assessment for the Import Tolerance on Fruiting Vegetables. D315531, Douglas Dotson, 5/ 9/ 2005. 2. Kasugamycin. Tolerance Petition Requesting Food Use of the Fungicide Kasugamycin on Imported Fruiting Vegetables ( Crop Group 8). Summary of Analytical Chemistry and Residue Data. Petition Number 3E6579. D308573, William T. Drew, 8/ 17/ 2005.

Appendices A 1.0 TOXICOLOGY DATA REQUIREMENTS The requirements ( 40 CFR 158.340) for food use ( tolerances on imported commodities only) of kasugamycin are in Appendix Table 1. Use of the new guideline numbers does not imply that the new ( 1998) guideline protocols were used.

APPENDIX TABLE 1 Toxicology Data Requirements.

Test Technical Required Satisfied 870.1100 Acute Oral Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . 870.1200 Acute Dermal Toxicity . . . . . . . . . . . . . . . . . . . . . . . 870.1300 Acute Inhalation Toxicity . . . . . . . . . . . . . . . . . . . . . 870.2400 Primary Eye Irritation . . . . . . . . . . . . . . . . . . . . . . . . 870.2500 Primary Dermal Irritation . . . . . . . . . . . . . . . . . . . . . 870.2600 Dermal Sensitization . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes yes yes 870.3100 Oral Subchronic ( rodent) . . . . . . . . . . . . . . . . . . . . . . 870.3150 Oral Subchronic ( nonrodent) . . . . . . . . . . . . . . . . . . . 870.3200 21 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.3250 90 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.3465 90 Day Inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes no no no yes yes no no no 870.3700a Developmental Toxicity ( rodent) . . . . . . . . . . . . . . . . 870.3700b Developmental Toxicity ( nonrodent) . . . . . . . . . . . . . 870.3800 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes APPENDIX TABLE 1 Toxicology Data Requirements.

Test Technical Required Satisfied Page 34 of 48 870.4100a Chronic Toxicity ( rodent) . . . . . . . . . . . . . . . . . . . . . 870.4100b Chronic Toxicity ( nonrodent) . . . . . . . . . . . . . . . . . . 870.4200a Oncogenicity ( rat) . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.4200b Oncogenicity ( mouse) . . . . . . . . . . . . . . . . . . . . . . . . 870.4300 Chronic/ Oncogenicity . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes 870.5100 Mutagenicity   Gene Mutation  bacterial . . . . . . . . . 870.5300 Mutagenicity   Gene Mutation  mammalian . . . . . . . 870.5375 Mutagenicity   Structural Chromosomal Aberrations . 870.5xxx Mutagenicity   Other Genotoxic Effects . . . . . . . . . . . yes yes yes yes no* yes no* yes 870.6100a Acute Delayed Neurotox. ( hen) . . . . . . . . . . . . . . . . . 870.6100b 90 Day Neurotoxicity ( hen) . . . . . . . . . . . . . . . . . . . . 870.6200a Acute Neurotox. Screening Battery ( rat) . . . . . . . . . . 870.6200b 90 Day Neuro. Screening Battery ( rat) . . . . . . . . . . . . 870.6300 Develop. Neuro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . no no no no no no no no no no 870.7485 General Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . 870.7600 Dermal Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . yes no yes no * Although these two mutagenicity studies were not classified as acceptable/ guideline, the requirement for these studies has been waived. See Section 4.1 for more details.

A 2.0 NON CRITICAL TOXICOLOGY STUDIES Executive summaries of the toxicology studies not used for toxicity endpoint selection or FQPA assessment are as follows.

A 2.1 Subchronic Oral Toxicity  Rat ( 870.3100) In a 90 day oral toxicity study ( MRID # 45910020), kasugamycin hydrochloride hydrate ( 64.5% ai; Lot # KP 834) was administered to 12 Wistar rats/ sex/ dose in the diet at dose levels of 0, 300, 1000, 3000, or 6000 ppm ( equivalent to 0/ 0, 17.5/ 20.3, 58.2/ 69.2, 176.7/ 201.0, and 354.8/ 395.5 mg/ kg bw/ day in males/ females, respectively). It was assumed, based on dose formulation concentration analyses, that the doses were adjusted for compound purity; however, this was not stated explicitly. No treatment related effects were observed on mortality, clinical signs, food consumption, food efficiency, water consumption, ophthalmoscopic examination, hematology, clinical chemistry, organ weights, or macroscopic pathology. There were indications of a slight systemic toxicity at 6000 ppm. At this dose, male body weights were significantly lower ( 4 9%) than controls ( p  0.05) each week starting on Week 2 until sacrifice, resulting in an 11% decrease in overall ( Weeks 0 13) body weight gain. In the 6000 ppm females, body weights were significantly lower ( 5%) than controls ( p  0.05) only on Week 6; however, non significant decreases in body weight gain were observed throughout the Page 35 of 48 study, resulting in a decrease of 8% in overall body weight gain. Although not considered treatment related, significant ( p  0.05) decreases in food consumption were seen in both sexes during the first week of dosing, indicating a possible palatability issue; average food consumption ( Weeks 0 13) was non significantly decreased by 4 6% in 6000 ppm males and females. An increased ( p  0.05) number of epithelial cells in the urinary sediment was observed in the females. Urine pH was decreased in both sexes; this is possibly due to the acidic properties of the test compound. Microscopically, in the 6000 ppm group, increased ( p  0.05) incidences were observed of eosinophilic bodies in the proximal tubular cells in the kidney of slight severity in the males ( 11/ 12 treated vs. 0/ 12 controls) and foam cell aggregation in lungs ( all of slight severity except 1 treated of moderate severity, 10/ 12 treated vs. 3/ 12 controls). These same lesions were found in the 3000 ppm group in the concurrently submitted combined chronic toxicity / carcinogenicity study ( MRID # 45910024), without any additional treatment related lesions in the organs to corroborate toxicity. Additionally, these lesions were graded slight ( lowest grade reported). While related to treatment, the increased incidence of both of these effects was not considered biologically significant. No adverse effects were observed at doses equal to or lower than 3000 ppm. Although an apparent decrease in water consumption was seen in the 3000 ppm males, mean water consumption data was based on individual cage values, and no baseline water consumption measurements were taken prior to dosing. Therefore, there is not enough information to make a definitive evaluation of a treatment related effect on male water consumption. Decreases in hematocrit, hemoglobin, and erythrocyte counts at 3000 and 6000 ppm were minor and not considered biologically significant. Increased incidences of increased eosinophilic bodies in the kidneys in males and foam cell aggregation in the lungs in the females were also not considered toxicologically significant. The LOAEL is 6000 ppm ( equivalent to 354.8/ 395.5 mg/ kg/ day in males/ females), based on decreased body weights and body weight gains in both sexes. The NOAEL is 3000 ppm ( equivalent to 176.7/ 201.0 mg/ kg/ day in males/ females). This study is classified acceptable/ guideline and satisfies the guideline requirements for a 90 day oral toxicity study ( OPPTS 870.3100a; OECD 408) in the rat.

A 2.2 Subchronic Oral Toxicity  Mouse ( 870.3100) In this subchronic oral toxicity study ( MRID # 45910019), 12 CD 1 mice/ sex/ dose were exposed to kasugamycin hydrochloride hydrate ( 64.8% ai; Batch #: KP 821) in the diet at nominal concentrations of 0, 300, 1000, 3000, or 10,000 ppm ( equivalent to 0/ 0, 41.2/ 58.0, 135.4/ 170.9, 408.5/ 565.6, and 1559/ 1834 mg/ kg/ day in males/ females) for up to 14 weeks. It was assumed, based on dose formulation concentration analyses, that the doses were adjusted for compound purity; however, this was not stated explicitly. No treatment related effects were observed on food or water consumption, ophthalmoscopic examination, hematology, clinical chemistry, urinalysis, or organ weights. At  3000 ppm, generally, from Week 4 until study termination, perianal reddening was observed in both sexes. Increased incidences of minimal to marked active chronic inflammation and ulceration of the anus was observed in both sexes. Increased mortality was also observed in both sexes. Four of the six decedent animals in the  3000 ppm groups were suffering from these lesions, and 3 of these 4 animals were killed because of extensive perianal skin lesions. Minimal to severe basophilia/ hyperplasia in the pars recta of the kidneys in females was also observed. Page 36 of 48 At 10,000 ppm, body weights were decreased generally throughout treatment in both sexes, as were overall ( Weeks 0 14) body weight gains and overall ( Weeks 1 14) food efficiency. In females, an increased incidence of emaciated appearance, perianal/ perigenital stain, and dark perianal regions were also observed. Increased incidences of testicular tubular dilatation, tubular degeneration associated with dilatation, and spermatoceles were observed. Additionally, extramedullary hematopoiesis in the spleen was also observed in the males. The LOAEL is 3000 ppm ( equivalent to 408.5/ 565.6 mg/ kg/ day in males/ females), based on increased mortality and anal lesions in both sexes, and kidney lesions in the females. The NOAEL is 1000 ppm ( equivalent to 135.4/ 170.9 mg/ kg/ day in males/ females). This study is classified as acceptable/ guideline and satisfies the guideline requirements ( OPPTS 870.3100a; OECD 408) for a subchronic oral toxicity study in the mouse.

A 2.3 Subchronic Oral Toxicity  Dog ( 870.3150) In a subchronic oral toxicity study ( MRID # 46030302), kasugamycin hydrochloride hydrate ( 80.6% ai; dose levels adjusted for purity; Lot No.: KP 913) was administered to 4 beagle dogs/ sex/ group in the diet at dose levels of 0, 300, 3000, or 6000/ 4500 ppm ( equivalent to 0/ 0, 11/ 11, 106/ 108, and 182/ 179 mg/ kg/ day [ M/ F], respectively) for 13 weeks. No treatment related effects were observed on mortality, ophthalmoscopy, hematology, urinalysis, or organ weight. At  3000 ppm, few feces, swollen mouth, excessive salivation, and thickened skin at the commissure of the mouth were observed in both sexes. Increased incidences of clinically observed tongue erosions/ ulcerations were observed in both sexes. Histopathological tongue lesions, including moderate to severe atrophy of the dorsal epithelium and moderate to severe loss of epithelial papillae of the dorsal surface, were observed in both sexes; and moderate to severe tongue ulcerations were observed in males. In the high dose group initially at 6000 ppm, tongue erosions/ ulcerations were clinically observed and food consumption during Weeks 1 through 5 was reduced ( 6 to  45%) in both sexes. Consequently, these animals were offered control diet between Weeks 6 ( Day 41) and 8 ( Day 50) and started on a reduced dietary dose of 4500 ppm from Week 8 through the end of the study. Discolored feces were observed at 6000/ 4500 ppm. Body weights were decreased ( p<= 0.05) in males during Weeks 6 9 ( 9 to  20%) and in females during Weeks 4 9 ( 13 to  27%). Cumulative body weight gain was decreased ( 56 to  109%; p  0.05) in males in Weeks 6 and 7 and in females from Weeks 4 to 8. Increased incidences of histopathological lesions of the tongue were observed in both sexes, including slight to marked serous exudate, minimal to severe ulceration, and minimal to slight chronic active inflammation. These lesions of the tongue appear to follow a consistent progression and likely contribute to decreased food consumption and decreased body weights. The only finding observed at 300 ppm was excessive salivation in females. The LOAEL is 3000 ppm ( equivalent to 106/ 108 mg/ kg/ day [ M/ F]) based on clinical signs ( tongue erosions/ ulcerations, few feces, swollen mouth, excessive salivation, and thickened skin at the commissure of the mouth), atrophy of the dorsal epithelium of the tongue, and loss of epithelial papillae of the dorsal surface of the tongue in both sexes. The NOAEL is 300 ppm ( equivalent to 11/ 11 mg/ kg/ day [ M/ F]). This study is classified as acceptable/ guideline and satisfies the guideline requirements ( OPPTS 870.3150; OECD 409) for a subchronic oral toxicity study in the dog. Page 37 of 48 A 2.4 Chronic Oral Toxicity  Dog ( 870.4100b) In a chronic oral toxicity study ( MRID # 46185901), kasugamycin hydrochloride hydrate ( 72.8% ai; dose levels adjusted for purity; Lot # KMB 0027) was administered to 4 beagle dogs/ sex/ group in the diet at dose levels of 0, 300, 1000, or 3000 ppm ( equivalent to 10.5/ 9.4, 30.5/ 33.4, 99.6/ 103.6 mg/ kg/ day [ M/ F], respectively) for 52 weeks. No adverse treatment related effects were observed on mortality, clinical signs, body weight, body weight gain, food consumption, food efficiency, ophthalmoscopy, hematology, organ weights, or gross or microscopic pathology. Gross and histopathological tongue lesions that were observed in both sexes in a concurrently submitted 13 week feed study ( MRID # 46030302) at doses of 3000 and 6000/ 4500 ppm were not observed in this study at 3000 ppm. The LOAEL was not observed. The NOAEL is 3000 ppm ( equivalent to 99.6/ 103.6 mg/ kg/ day [ M/ F]), the highest dose tested in the study. Although the investigators did not test to the limit dose and a LOAEL was not observed, the doses selected were appropriate based on results from a 13 week subchronic study in dogs in which tongue lesions were observed at 3000 ppm and 6000/ 4500 ppm that affected food consumption and body weight. This study is classified as acceptable/ guideline and satisfies the guideline requirements ( OPPTS 870.4100b; OECD 452) for a chronic oral toxicity study in the dog.

A 2.5 Carcinogenicity  Mouse ( 870.4200b) In a carcinogenicity study ( MRID # 46030304), kasugamycin hydrochloride hydrate ( 64.5 64.9% ai, dose levels adjusted for purity, Lot # KP 821) was administered to 72 CD 1 mice/ sex/ group in the diet at dose levels of 0, 50, 300, or 1500 ppm ( equivalent to 0/ 0, 5.93/ 7.25, 34.94/ 42.49, and 186.3/ 215.2 mg/ kg/ day [ M/ F], respectively) for up to 78 weeks, with an interim sacrifice of twenty mice/ sex/ group at Week 52. No adverse treatment related effects were observed on mortality, body weight, body weight gain, food consumption, food efficiency, hematology, organ weights, gross pathology, or microscopic ( non neoplastic or neoplastic) findings. The LOAEL was not observed. The NOAEL is 1500 ppm ( equivalent to 186.3/ 215.2 mg/ kg/ day [ M/ F]), the highest dose in the study. Although the investigators did not test to the limit dose and a LOAEL was not observed, the doses selected were appropriate based on results from a 13 week subchronic oral toxicity study in which mice were dosed at 0, 300, 1000, 3000, or 10,000 ppm ( MRID # 45910019). In the subchronic study, increased mortality and histopathological changes in the kidney ( basophilia/ hyperplasia in the pars recta) and anus ( chronic active inflammation, ulceration, and perianal reddening) were observed at 3000 ppm. Under the conditions of this study, the carcinogenic potential of kasugamycin hydrochloride hydrate is negative. A sufficient maximum tolerated dose ( MTD) was not achieved in this study. However, kasugamycin hydrochloride hydrate was negative for mutagenicity in concurrently reviewed studies, including the V79/ HGPRT forward mutation assay ( MRID # 45910026), a cytogenetic assay for chromosome aberrations in CHO cells ( MRID # 45910025), a bacterial gene mutation and DNA damage assay ( MRID # 45910028), an in vivo micronucleus assay in mice ( MRID # 46030305), and a study for unscheduled DNA synthesis ( MRID # 45910027). Additionally, the carcinogenic potential of kasugamycin hydrochloride hydrate was negative in a concurrently reviewed 24 month oral chronic toxicity and carcinogenicity study in Page 38 of 48 rats ( MRID # 45910024). This study is classified as acceptable/ guideline and satisfies the guideline requirements ( OPPTS 870.4100; OECD 452) for a chronic oral toxicity study in the mouse.

A 2.6 Mutagenicity  Bacterial Gene Mutation ( 870.5100) and Bacterial DNA Damage ( 870.5500) In this study ( MRID # 45910028), three different bacterial assays were performed. In a DNA damage assay, Bacillus subtilis strains H17 and M45 were exposed overnight to kasugamycin HCl ( purity: 80% ai; Lot/ Batch #: not reported) in distilled water at concentrations of 0, 20, 100, 200, 1000, or 2000

g/ disk. The diffusion test method was used. In an in vitro gene mutation assay, S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 and E. coli strain WP2hcr were exposed to kasugamycin HCl in distilled water at concentrations of 0, 5, 10, 50, 100, or 200

g/ plate in the presence and absence of S9 activation. Additionally, S. typhimurium strain G46 ( his) was similarly tested at concentrations of 0, 5, 10, 50, 100, or 500

g/ plate in the absence of S9 activation only. Strain specific positive controls were used. In a non guideline host mediated gene mutation assay, 5 6 male ICR mice/ dose received two equal doses ( gavage, 20 mL/ kg) of kasugamycin HCl in distilled water over a 24 hour period at doses of 500 or 2000 mg/ kg ( total dose received was 1000 or 4000 mg/ kg). Immediately after the second dosing, a 2 mL suspension of S. typhimurium strain G46 ( his) was inoculated via i. p. injection. Three hours after the final treatment, the peritoneal fluid was removed and used in an Ames reverse mutation assay. In the DNA damage assay, kasugamycin HCl was tested up to 2000

g/ disk. No treatment related increases in the difference between zones of inhibition were observed in any treatment group compared to the concurrent controls. The positive controls induced the appropriate response. There was no evidence that DNA damage was induced. The study is classified as acceptable/ guideline and satisfies the guideline requirements ( OPPTS 870.5500) for other genotoxic mutagenicity ( bacterial DNA damage) data. In the gene reverse mutation assay, kasugamycin HCl was tested up to 500

g/ plate in S. typhimurium strain G46 ( his) in the absence of S9, and up to 200

g/ plate in all other strains (+/ S9). It was not reported if any evidence of cytotoxicity ( thinning of the background lawn or reduction in number of revertants) was observed. No treatment related increases in the mean number of revertants/ plate were observed in any bacterial strain at any dose level in the presence or absence of S9 activation compared to solvent controls. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background in the presence or absence of S9. Because the test material was not evaluated at the limit dose ( 5000

g/ plate); there was no indication of test material induced cytotoxicity; and there was no defined limit of solubility, it could not be determined if a sufficiently high dose was used. Therefore, the study is classified as unacceptable/ upgradable and does not satisfy the guideline requirements ( OPPTS 870.5100; OECD 471) for in vitro mutagenicity ( bacterial reverse gene mutation). The study may be upgraded to acceptable pending submission of data that indicate that the high dose was sufficient ( e. g.. test material solubility data or cytotoxicity data). In the host mediated assay, kasugamycin HCl was tested up to 4000 mg/ kg ( total dose). No significant increases in the mean mutation frequency were observed at either dose compared to controls. The positive control induced the appropriate response. There was no evidence of induced mutant colonies over background. The study is classified as acceptable/ nonguideline Page 39 of 48 A 2.7 Mutagenicity  Mammalian Gene Mutation ( 870.5300) In three independent mammalian cell gene mutation assays at the HGPRT locus ( MRID # 45910026) in the presence of S9 activation and two independent studies in the absence of S9 activation, Chinese hamster V79 lung fibroblasts cultured in vitro were exposed to kasugamycin hydrochloride hydrate ( 67.1% ai; Lot # KP 570) in Eagle's minimal essential medium at concentrations of 0.5, 1, 2, 4, 6, 8, or 10 mg/ mL for 4 hours. Kasugamycin hydrochloride hydrate was tested up to cytotoxic concentrations ( 10 mg/ mL [ Trial 1,  S9 and Trial 2, + S9] and >= 8 mg/ mL [ Trial 1, + S9]). No treatment related increases in mutant frequency were observed in any trial in the presence or absence of S9. The positive controls induced the appropriate response. There was no evidence that kasugamycin hydrochloride hydrate induced mutant colonies over background in the presence or absence of S9 activation. This study is classified as acceptable/ guideline and satisfies the guideline requirements ( OPPTS 870.5300, OECD 476) for in vitro mutagenicity ( mammalian forward gene mutation) data.

A 2.8 Mutagenicity  Structural Chromosomal Aberrations ( 870.5375) In a mammalian cell cytogenetics assay ( chromosome aberration; MRID # 45910025), Chinese hamster ovary cell cultures were exposed to kasugamycin hydrochloride hydrate ( 67.1% ai; Lot # KP 570) in McCoy's 5a medium at concentrations of 1, 2, 3, 4, or 5 mg/ mL for 2 hours in the presence of S9 or for 7.25 hours in the absence of S9. Cells were harvested at 8 or 2.5 hours after conclusion of treatment in the presence or absence of S9, respectively. Kasugamycin hydrochloride was tested up to 5 mg/ mL (+/ S9). Although only evidence of slight cytotoxicity was observed at 5 mg/ mL (+/ S9) in the cytogenetic assay, the results of the preliminary study indicate that the dose levels selected for the main study were acceptable. There were no significant increases in the percentage of cells with aberrations ( excluding gaps) at any dose in the presence or absence of S9. The most frequently observed types of aberration ( excluding gaps) were acentric fragments and dicentrics. The positive controls induced the appropriate response. There was no evidence that kasugamycin hydrochloride induced a clastogenic effect in the presence or absence of S9 activation. However, the current guidelines recommend a harvest time of 1.5 cell cycles after treatment. The cell cycle for CHO cells is approximately 12 14 hours. The cells were harvested at only 10 hours after treatment, and there was evidence of a slight cell cycle delay at 5 mg/ mL in the absence of S9. Therefore, the reviewers conclude that the time from treatment to cell harvest was insufficient. This study is classified as unacceptable/ not upgradable and does not satisfy the guideline requirement ( OPPTS 870.5375, OECD 473) for in vitro mutagenicity ( chromosome aberration) data.

A 2.9 Mutagenicity  Mammalian Erythrocyte Microcnucleus ( 870.5395) In a bone marrow micronucleus assay ( MRID # 46030305), 5 non fasted CD 1 mice/ sex/ dose were treated once via gavage ( 15 mL/ kg) with kasugamycin hydrochloride hydrate ( 67.1% ai; Lot # KP 570) in distilled water at doses of 0, 200, 1000, or 5000 mg/ kg bw, and bone marrow cells were harvested at 24 hours post dosing. Additionally, 5 mice/ sex/ sacrifice time were similarly dosed at 0 or 5000 mg/ kg, and bone marrow cells were harvested at 48 or 72 hours post dosing. Chlorambucil ( 30 mg/ kg) served as the positive control. Kasugamycin hydrochloride hydrate was tested up to 5000 mg/ kg; however, no evidence of bone marrow Page 40 of 48 toxicity ( decreased PCE: NCE) was observed at any dose at 24, 48, or 72 hours post dosing. Clinical signs of toxicity were limited to brown anal staining and diarrhea at 5000 mg/ kg on the day of dosing. No significant increase in micronucleated polychromatic erythrocytes ( MPCEs) was observed at 24, 48, or 72 hours post dosing. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow compared to controls. The study is classified as acceptable/ guideline and satisfies the guideline requirement ( OPPTS 870.5395; OECD 474) for in vivo cytogenetic mutagenicity data.

A 2.10 Mutagenicity  Unscheduled DNA Synthesis in Mammalian Cells in Culture ( 870.5550) In three independent trials of an unscheduled DNA synthesis assay ( MRID # 45910027), HeLa S3 cell cultures were exposed to kasugamycin hydrochloride hydrate ( 67.1% ai; Lot No.: KP 570) in arginine free Eagle's Minimal Essential Medium for 3 hours at concentrations of 0.156, 0.313, 0.625, 1.25, or 2.50 mg/ mL (+/ S9; Trial 1) and 0.313, 0.625, 1.25, 2.50, 5.00, or 10.0 mg/ mL (+/ S9; Trial 2 and + S9; Trial 3). Kasugamycin hydrochloride hydrate was tested up to cytotoxic concentrations in the presence of S9 ( 10 mg/ mL, Trial 3). Although no evidence of cytotoxicity ( decreased amount of DNA recovered) was observed at up to 10 mg/ mL in the absence of S9, the results of the preliminary cytotoxicity test indicate that 10 mg/ mL was the appropriate high dose for the UDS assay. There were no reproducible increases (> 50% higher than the controls) in the incorporation of [ 3H] thymidine in the presence or absence of S9 at any dose. The positive controls induced the appropriate response in all trials. There was no evidence that unscheduled DNA synthesis, as determined by liquid scintillation counting procedures, was induced. The study is classified as acceptable/ guideline and satisfies the guideline requirement ( OPPTS 870.5550; OECD 482) for other genotoxic mutagenicity data.

A 2.11 Metabolism  Rat ( 870.7485) In a rat metabolism study ( MRID # 46030306), [ 14C] kasugamycin hydrochloride hydrate in sterile deionized water was administered to Fischer 344 ( F344 NHLa CFV) rats by gavage. In the main ADME study, 5 rats/ sex/ dose were given [ 14C] kasugamycin hydrochloride hydrate ( Batch # CP 1916; radiochemical purity > 98%) either as a single oral dose of 100 or 1000 mg/ kg, or as a repeated oral dose of 100 or 1000 mg/ kg ( 14 days unlabeled + 1 day radiolabeled). Pharmacokinetic, tissue distribution time course, and biliary excretion studies were also performed on males and females at both dose levels. Metabolites were identified and quantified in the urine and feces from the main ADME study and in plasma, kidney, and liver from the tissue distribution time course study. In the main ADME study, the total recovery of the radioactive dose ranged from 90.7 96.8% dose at 168 hours post dose, with the majority of the dose recovered within 48 hours in the feces ( 81.9 93.9%) and urine ( 1.26 3.07%). Fecal excretion accounted for 87.7 94.5% in all groups; urinary excretion accounted for 1.35 3.26%; and the cage rinse, wash, and wipe accounted for <= 1.14%. The carcass/ tissues retained <= 0.13% dose, and results from a previous study indicated a minimum amount of 14CO 2 (< 0.2% of total radioactivity) was detected in exhaled air. For the bile duct cannulated rats, < 0.01% of the dose was recovered in the bile. For both sexes, concentrations of radioactivity in plasma peaked within 1 hour of dosing Page 41 of 48 for both the 100 mg/ kg dose group ( 1.47 2.17

g/ g) and the 1000 mg/ kg dose group ( 5.23 6.40

g/ g) and declined to below the limit of quantitation by 24 hours post dose. A terminal half life of radioactivity of 1.17 1.55 hours was observed in plasma. The total dose absorbed as indicated by the area under the curve ( AUC) was proportional to dose in both sexes at both 100 mg/ kg ( 2.88 3.63

g* hour/ g) and 1000 mg/ kg ( 15.3 18.7

g* hour/ g). The time course of tissue distribution showed that the concentration of radioactivity was higher in the kidneys, urinary bladder, and lymph nodes than in blood in both sexes at all time points in both the low ( 0.582 30.3

g Eq/ g vs <= 1.80

g Eq/ g in blood) and high ( 6.43 147.0

g Eq/ g vs <= 8.32

g Eq/ g in blood) dose groups. In the pancreas, increases over blood levels were also observed in the low and high dose females at all time points and in the low and high dose males at selected time points. Concentrations of radioactivity were also approximately twice as high in plasma compared to whole blood. All other tissues ( excluding G. I. tract) had concentrations of radioactivity that were comparable to or below the concentrations in the blood throughout the time course. By 168 hours post dose, concentrations of radioactivity were nondetectable in all but a few tissues, with the highest concentrations being detected in kidneys ( 1.89 24.7

g Eq/ g) in both sexes and in all groups. Radioactivity was also detected in the intestinal tract ( 0.92 1.11

g Eq/ g) and stomach ( 1.14 1.21

g Eq/ g) of both sexes from the 1000 mg/ kg single dose group, and in the intestinal tract ( 0.48

g Eq/ g) of females from the 1000 mg/ kg repeated dose females. The metabolism of kasugamycin hydrochloride hydrate in rats was not affected by sex, dose level, or duration of dosing. Parent was the major component identified in the urine, feces, liver, kidney and plasma, and minor amounts (< 1% dose) of the metabolite kasuganobiosamine were identified in urine, liver, kidney and plasma. The presence of kasugamycinic acid was also postulated in liver extracts. Parent compound was identified by TLC and HPLC; metabolites were identified by mass spectroscopy. In the main ADME study ( Groups A D), parent was the predominant compound identified in both feces ( 79.9 89.8% dose) and urine ( 1.3 2.9% dose) in all groups, totaling 81.6 92.7% dose. In the urine from all groups, kasuganobiosamine was identified by mass spectroscopy, but was not quantified as the metabolite could not be resolved from the parent by HPLC. Kasuganobiosamine was not detected in the feces. Parent and kasuganobiosamine were also detected in the kidney and liver extracts from animals sacrificed 1 6 hours post dose, and kasugamycinic acid was also putatively detected in the liver. Radioactivity in the kidney and liver accounted for < 0.01 0.18% of the dose in these samples and  0.03% of the dose in animals sacrificed at 168 hours post dose. Parent and kasuganobiosamine were also reportedly detected in plasma. Regardless of sex, dose level ( 100 or 1000 mg/ kg), and duration of dosing, the absorption and metabolism of [ 14C] kasugamycin hydrochloride hydrate in rats was limited (< 5% dose). This metabolism study in the rat is classified acceptable/ guideline and satisfies the guideline requirement for a Tier 1 metabolism study [ OPPTS 870.7485, OPP 85 1] in rats.

A 3.0 METABOLISM CONSIDERATIONS A 3.1 Team Proposal The only metabolite which would possibly be considered for inclusion in the ROC ( in addition to the parent) was kasugamycinic acid, which was present at less than 10% in all foliage Page 42 of 48 samples, and only occurred at concentrations of 10% or more in fruit samples from the 21 and 28 day PHIs. Because the petitioner is proposing a PHI of 1 day on imported fruiting vegetables, kasugamycinic acid is unlikely to occur as other than a minor constituent in imported fruiting vegetable commodities, and was therefore not included in the ROC for either tolerance expression or risk assessment purposes. Also, as residues of kasugamycin in all samples from the crop field trials ( at 1X) were either non detectable or less than the LOQ, the increase in risk from inclusion of this relatively minor metabolite was expected to be negligable.

A 3.2 Nature of the Residue Studies in Plants Executive Summary of Tomato Metabolism Study Arvesta Corporation, on behalf of Hokko Chemical Industry Company, Limited ( Japan), has submitted a study investigating the metabolism of [ 14C] kasugamycin in tomatoes. The radiolabeled test substance was applied as a single foliar broadcast spray to tomato plants at approximately 0.17 pound active ingredient per acre ( lb ai/ A), slightly more than 3X the proposed maximum seasonal application rate. Mature tomatoes and foliage were harvested 2 hours and 1, 7, 14, 21, and 28 days following application. Residues were higher in the tomato foliage than in the fruit. TRR, determined by summing extractable and nonextractable radioactivity, were 0.011, 0.008, 0.027, 0.072, 0.098, and 0.084 ppm in tomatoes harvested 2 hours, 1 day, 7 days, 14 days, 21 days, and 28 days after treatment, respectively. TRR in foliage from the respective pre harvest intervals ( PHIs) were 2.86, 4.73, 1.77, 2.92, 2.57, and 4.29 ppm. Extraction with water removed 91 94% of the TRR from fruit and 80 86% TRR from foliage. Extraction with methanol released an additional 2 3% TRR from fruit with 14 through 28 day PHIs and 4 8% TRR from the corresponding foliage. Nonextractable residues accounted for a maximum of 9% TRR in fruit and no more than 12% TRR in foliage. Residues were characterized or identified primarily by HPLC analysis, with confirmatory analysis by TLC. These methods successfully identified the predominant residues in tomatoes and foliage. Approximately 90% and 94% of the TRR were identified in tomatoes harvested 2 hours and 1 day after treatment, respectively, and 55 69% TRR were identified in tomatoes harvested 7 28 days after harvest. In foliage, roughly 86% and 80% TRR were identified in samples harvested 2 hours and 1 day after treatment, respectively, and 60 72% TRR were identified in foliage from the remaining sampling intervals. In general the metabolite profile was similar in tomato fruit and foliage. Parent compound ( kasugamycin per se) was the major identified component in all samples from all harvest intervals, accounting for 90.4 93.9% TRR in tomatoes from the 2 hour and 1 day PHIs, for 69.2% TRR in tomatoes from the 7 day PHI, and for 54.8 59.5% TRR in tomatoes from the remaining sampling intervals. In foliage, kasugamycin accounted for 75.0 84.0% TRR in samples from the 2 hour and 1 day PHIs, for 69.5% TRR in samples from the 7 day PHI, and for 52.5 57.2% TRR in foliage from the remaining sampling intervals. In tomatoes, the only other identified metabolite was kasugamycinic acid, which was found in tomatoes from the 21 and 28 day PHIs at 9.5% and 12.0% TRR, respectively. Kasugamycinic acid was also identified in foliage from all harvest intervals at 2.4 7.1% TRR. Two additional metabolites were identified in foliage: 2 N acetyl kasugamycin was identified in 14 through 28 day PHI foliage at 0.7 2.2% TRR, and kasuganobiosamine was identified in 1 through 28 day PHI tomato foliage at 0.4 1.0% TRR. Remaining radioactivity was characterized as unknowns, each present at maximum concentrations of 19.5% TRR in fruit and 12.2% TRR in foliage. Unknowns accounting for 13.5 19.5% TRR in fruit and 2.9 12.2% TRR in foliage were subjected to further Page 43 of 48 characterization procedures which confirmed that they were comprised of multiple minor components.

Based on the results of the tomato metabolism study the petitioner proposes that the major metabolic pathway of kasugamycin in tomatoes involves conjugation of the parent compound, conversion to kasugamycinic acid, and subsequent conjugation of kasugamycinic acid. Conversion of kasugamycin to 2 N acetyl kasugamycin and kasuganobiosamine was thought to be a minor metabolic route. Page 44 of 48 Tabular Summary of Tomato Metabolism Study APPENDIX TABLE 2 Summary of Characterization & Identification of Radioactive Residues in Tomato Fruit Following Foliar Broadcast Application of [ 14C] Kasugamycin at 0.17 lb ai/ A.

Compound 2 hour PHI 1 Day PHI 7 Day PHI 14 Day PHI 21 Day PHI 28 Day PHI TRR = 0.011 ppm TRR = 0.008 ppm TRR = 0.027 ppm TRR = 0.072 ppm TRR = 0.098 ppm TRR = 0.084 ppm % TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR ppm Kasugamycin 90.4 0.010 93.9 0.007 69.2 0.018 54.8 0.039 59.5 0.058 57.3 0.049 Kasugamycinic acid         9.5 0.009 12.0 0.010 Unknown 1       3.2 0.002

Unknown 2     13.5 0.004 19.5 0.014 15.6 0.015 14.1 0.012 Unknown 3     5.5 0.001 7.7 0.006 5.5 0.005 7.1 0.006 Unknown 4     4.9 0.001 3.4 0.002

Others       2.3 0.002

Methanol extractable       2.8 0.002 3.1 0.003 2.7 0.002 Total identified 90.4 0.010 93.9 0.007 69.2 0.018 54.8 0.039 69.0 0.067 69.3 0.059 Total characterized     23.9 0.006 38.9 0.028 24.2 0.023 23.9 0.020 Total extractable 91.0 0.010 94.1 0.007 93.7 0.025 93.1 0.067 93.7 0.092 93.5 0.079 Unextractable ( PES) 9.0 0.001 5.9 < 0.001 6.3 0.002 6.9 0.005 6.3 0.006 6.5 0.005 Accountability* 100 100 100 100 100 100 * Accountability = Total Extractable + Total Unextractable. Accountabilities were 100% because the TRR were determined by summing extractable and nonextractable residues. Page 45 of 48 APPENDIX TABLE 3 Summary of Characterization & Identification of Radioactive Residues in Tomato Foliage Following Foliar Broadcast Application of [ 14C] Kasugamycin at 0.17 lb ai/ A.