Kasugamycin Human Health Risk Assessment DP Barcode 0376608



### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, DC 20460

OFFICE OF

CHEMICAL SAFETY AND

POLLUTION PREVENTION



### MEMORANDUM

DATE: 

### 13 July 2012

SUBJECT: 

### Kasugamycin. Human Health Risk Assessment for the Proposed Use of the Fungicide on Fruiting Vegetables, Po me Fruits, and Walnuts.

PC Code: 230001

Decision Number: 427886

Petition Number: OF7689
Risk Assessment Type: Single Chemical Aggregate

TXR Number: NA

MRID Numbers: NA

FROM:

THROUGH:



### To: Shaunta Hill, RM Team 21 Cynthia Giles

- Parker

, Chief Fungicide Branch (FB) Registration Division (RD), 7505P DP Barcode: 0376608 Registration Number: 66330-404 Regulatory Action: Section 3 Registration

Case Number: 7045

CAS Number: 6980

- 18

- 3

40CFR: § 180 .614

Page 1 of60Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 2 of 60 Table of Contents

2.0 HED Recommendations ................................................................................................... 5

2.2 Tolerance Considerations .............................................................................................. 6

2.2.1 Enforcement Analytical Method ............................................................................... 6

2.2.2 International Harmonization ..................................................................................... 6

2.2.3 Recommended Tolerances ......................................................................................... 7

2.2.4 Revisions to Petitioned-for Tolerances ..................................................................... 8

2.3 Label Recommendations ................................................................................................ 8

2.3.1 Recommendations from Re sidue Chemistry Reviews ............................................. 8

3.5 Consideration of Environmental Justice .................................................................... 12

4.0 Hazard Characterization and Dose-Response Assessment ......................................... 12

4.1 Toxicology Studies Available for Analysis ................................................................. 13

4.2 Absorption, Distribution, Metab olism, & Elimination (ADME) ............................. 13

4.2.1 Dermal Absorption ................................................................................................... 14

4.3 Toxicological Effects .................................................................................................... 14

4.4 Safety Factor for Infants and Children (FQPA Safety Factor) ............................... 16

4.4.1 Completeness of the Toxicology Database .............................................................. 17

4.4.2 Evidence of Neurotoxicity ........................................................................................ 17

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal ........ 17

4.4.4 Residual Uncertainty in the Exposure Database ................................................... 17

4.5 Toxicity Endpoint and Point of Departure Selections .............................................. 17

4.5.1 Dose-Response Assessment ...................................................................................... 17

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment ..... 19

4.5.3 Cancer Classification and Risk Assessment Recommendation ............................ 19

4.5.4 Summary of Points of Departure and T oxicity Endpoints Used in Human Risk

Assessment

.............................................................................................................................. . 19

A.3 Hazard Identification and Endpoint Selection .......................................................... 43

A.4 Executive Summaries (Studies Submit ted since Previous Assessments) ................. 45Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 3 of 60 1.0 Executive Summary

Arysta LifeScience North America LLC, as an agent for Hokko Chemical Industry Company Limited (Japan), has proposed the establishment of permanent tolerances for residues of the aminoglycoside fungicide kasugamycin (3- O -[2-amino-4-[(carboxyimino-methyl)amino]- 2,3,4,6-tetradeoxy- α -D- arabino -hexopyranosyl]-D- chiro -inositol, CAS #6980-18-3) in or on fruiting vegetables, pome fruits, and walnuts. The proposal is associat ed with the first US registration for this active ingredient (ai).

Kasugamycin is a low-use-rate, wide-spectru m aminoglycoside antibiotic fungicide produced

from Streptomyces kasugaensis . Kasugamycin formulations are applied as foliar sprays in agriculture and horticulture. Ka sugamycin has preventative, curative and systemic activity at low application rates. The compound controls a range of phytopathogenic organisms in the proposed crops. Kasugamycin is currently used in over 20 countries on different crops including tomato, paprika, eggplant, rice and potato. HED previously recommended in favor of a tolerance for residues of kasugamycin in imported fruiti ng vegetables and a time-limited tolerance for residues in apples.

Kasugamycin is a member of the aminoglycosid e family of antibiotics, which also includes streptomycin and gentamicin. These agents inhi bit bacterial protein synt hesis by binding to the

30S subunit of the bacterial ribosome. Their penetration through the cell membrane of the bacterium depends partly on oxygen-dependent ac tive transport by a poly amine carrier system that seems to be absent in mammalian systems. This risk assessment document does not address the potential impact of the pest icidal use of kasugamycin on an tibiotic resistance. Tolerance

levels as recommended within this document are presented excluding antibiotic resistance impacts/concerns.

The toxicology database is comple te for kasugamycin, and the available guideline toxicity data are considered adequate for risk assessment purpos es. Kasugamycin exhibits low acute toxicity. The major effects observed across species in mu ltiple-dose studies were decreased body weights and body weight gains. Testicul ar atrophy was also observed in the rat reproductive and mouse subchronic toxicity studies, but only at doses significantly hi gher than the lowest-observedadverse-effect level (LOAEL) in the rat study, or above the limit dose in the mouse. There was no evidence that exposure to kasugamycin re sults in neurotoxicity, and a developmental neurotoxicity (DNT) study is not required. A 28- day rat inhalation toxici ty study has not been submitted. However, HED’s Hazard and Science Policy Council (HASPOC) has determined that a repeated exposure inhalation study is not requir ed, based on the available hazard and exposure information, including the following considerations: sel ection of irritation as the endpoint of concern for inhalation exposure, based on irritation seen following oral exposures in multiple studies; the use of an oral no-observed-adve rse-effect level (NOAEL) which results in occupational handler margins of exposure (M OEs) ranging from 58,000 to 2,500,000 for risk via the inhalation route; the physicochemical propertie s, and low volatility, of kasugamycin; the lowKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 4 of 60 acute inhalation toxicity (Toxici ty Category IV); and the low pot ential for inhalation exposure, based on the proposed use pattern (TXR #0052461).

Because of kasugamycin’s toxicological profile, lo w application rates, low vapor pressure, lack of residential uses, lack of persistence in the environm ent, and inhalation MOEs all ≥ 58,000, a

10X UF DB is not required for the lack of the inhalation study.

The database is complete with respect to pre- and post-natal toxicity, an d shows no evidence of increased qualitative or quantitative susceptibility in the offspring, or in the developing fetus. Based on the hazard and exposure data, HED has recommended that the required Food Quality Protection Act (FQPA) 10X safety factor be reduced to 1X. Ther efore, uncertainty factors used

in the assessment consist of a 10X factor for interspecies extrapolati on, and a 10X factor for intraspecies variability. There was no evidence of carcinogenicity in male and female mice, nor

in male and female rats at doses that were adequate to assess th e carcinogenic potential of kasugamycin. There was no evidence of mutage nicity. Based on the overall weight of the evidence, kasugamycin is classified as "not likely to be carcinoge nic to humans”.

There were no toxic effects associated with a si ngle dose of kasugamycin; therefore, an acute dietary endpoint and dose were not selected for the general US population, or any population subgroup. The dose and endpoint from the rat co mbined chronic toxicity/carcinogenicity study were selected for chronic dietary exposure and risk assessment. The NOAEL of 11.3 mg/kg/day was based on testicular softening and atrophy at the LOAEL of 116 mg /kg/day. Testicular atrophy is consistent with fi ndings of other studies.

There was no systemic toxicity associated with dermal exposure in an acceptable dermal toxicity study in the rat, and therefore a dermal endpoint and dose were not selected. For inhalation exposure (short- and intermediate-term), the dog subchronic and rabbit de velopmental toxicity

studies were selected as co-c ritical studies. The endpoint of concern is irritation since kasugamycin appears to be irrita ting to the oral and gastrointes tinal tract mucosa following oral exposures. In the subchronic study with dogs, irrita tion of the oral cavity was characterized as tongue lesions, swollen mouth, excessive salivation, and thickening of the skin at the

commissures of the mouth. The NOAEL wa s 11 mg/kg/day, and the LOAEL was 106 mg/kg/day. The developmental rabbit study was selected as the co-critical study since the maternal NOAEL (10 mg/kg/day) was comparab le to the NOAEL in the dog study, and the maternal toxicity may have manifested due to the irritation pote ntial of the compound. The use of the oral NOAEL results in MOEs ranging fr om 58,000 to 2,500,000 for risk from occupational exposure via the inhalation route.

The nature of kasugamycin residues in plants and livestock (ruminants) is adequately

understood. The residue of concern for tolerance enforcement and risk assessment is the parent compound, although no tolerances are needed in livestock commodities based on the proposed uses. The parent kasugamycin and 2 degradates with similar mobility were recommended forKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 5 of 60 inclusion in the drinking water modeling. Adequa te residue data were submitted to support the proposed uses on fruiting vegeta bles, pome fruits, and walnuts. HED has used conservative assumptions to assess the risk from chronic dietary exposure to kas ugamycin through food and drinking water. These include the use of recommended tole rance-level residues, modeled drinking water estimates, and the assumption that all requested commodities are treated, or 100% crop treated (CT). Chronic diet ary risk estimates for kasugamy cin are below HED’s level of concern (LOC) of 100% of the chronic population adjusted do se (cPAD) for all population subgroups. The most highly exposed population subgroup, children 1-2 years old, had a risk

estimate of 1.7% cPAD.

There are no existing or propos ed residential uses of kas ugamycin. Therefore, the only aggregate scenario assessed was chronic aggregate risk, equivalent to chr onic dietary risk, which

is not of concern.

Occupational handler exposures to kasugamy cin were estimated assuming the maximum application rate, and label-reco mmended equipment and methods, a nd standard assumptions with respect to the area treate d. In the absence of chemical-speci fic data to assess handlers’ exposure and risk, HED relied on surrogate unit exposure data to estimate e xposure and risk. HED assumed a single layer of clothing (baseline), w ithout additional personal protective equipment (PPE). The resulting handler risk estimates, or MOEs, are all above 100, and are thus below HED’s LOC. Depending on the scenario, MOEs ranged from 58,000 to 2,500,000.

Although a quantitative occupational post-applic ation inhalation exposure assessment was not performed, an inhalation exposure assessment was performed for occupatio nal handlers. This

assessment resulted in risk estimates that did not exceed HED’s LOC at baseline inhalation PPE. Handler exposure resulting from application of pe sticides outdoors is likely to result in higher exposure than post-application exposure. Therefore, it is expected that these handler inhalation exposure estimates would be protective of o ccupational post-applicati on inhalation exposure

scenarios.

Review of human research: This risk assessment relies in part on data from studies in which adult human subjects were intent ionally exposed to a pesticide or other chemical. These studies (listed in Appendix D) have been determined to require a review of thei r ethical conduct, have received that review, and have been de termined to be ethically conducted.

2.0 HED Recommendations

HED recommends in favor of registration and establishment of tolerances for the use of kasugamycin on fruiting vegetables, pome fru its, and walnuts, as described below.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 6 of 60 2.1 Data Deficiencies

None.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

Adequate analytical methods are available fo r the purpose of tolerance enforcement.

In conjunction with the establishment of tolerances wit hout US registration (PP#3E6579), the petitioner proposed a reverse phase, ion pair ing method utilizing high-performance liquid chromatography with ultraviolet detection (HPLC/UV) for the en forcement of tolerances for residues of kasugamycin in plant commodities. The method is entitled Determination of Kasugamycin in Tomatoes, Potatoes, and Peppers (Morse Laboratories Method #Meth-146). The enforcement method for kasugamycin residues , as originally proposed, was forwarded to the Biological and Economic Analysis Division’s (BEAD’s) Analyti cal Chemistry Branch (ACB) for a petition method validation (PMV) trial. The method was subsequently modified as recommended by ACB, and was then deemed acceptable as an enforcement method.

The HPLC/UV enforcement method (Meth-146, Revi sion #4) was used to determine residues of kasugamycin in the studies associated with th e proposed uses. Samples were analyzed for residues of kasugamycin using laboratory working methods (FEQL Project #0407 for apple commodities, and FEQL Project #0706 for pears) which were adapted from Method #Meth-146, Revision #4, and included minor modifications to improve the performance of the methods. The methods were adequately validated both prior to , and in conjunction with, the analysis of the field trial and processing study samples. The lowest level of method validation (LLMV) was 0.040 ppm in each matrix tested.

2.2.2 International Harmonization

There are no established or pr oposed Codex or Canadian maximum residue limits (MRLs) for kasugamycin in any food or feed crops. Tole rances were established in 40CFR §180.614 at 0.04 ppm to cover residues in/on imported tomatoes a nd peppers from Mexico. There are no issues regarding compatibility of the proposed tolerances because Mexico adopts US tolerances, and/or Codex MRLs, for its export purposes. The curre ntly proposed uses are also under review in

Canada’s Pest Management Regulatory Agency (PMRA) ; both EPA and PMRA have agreed to harmonize recommended tolerances, based on th e use of the OECD to lerance calculation procedure.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 7 of 60 2.2.3 Recommended Tolerances

The tolerance expression should be revised to reflect specific coverage and compliance

statements for the toleran ces to be properly enforced in accordance with the Federal Food, Drug and Cosmetic Act ( FFDCA). Therefore, the tolerance expression for kasugamycin cited in 40CFR §180.614[a] should be revised to state:

Tolerances are established for residues of kasugamycin, including its metabolites and degradates, in or on th e commodities listed in the table below. Compliance with the tolerance levels specified below is to be determined by measuring only kasugamycin (3- O -[2-amino-4- [(carboxyimino-methyl)ami no]-2,3,4,6-tetradeoxy- α -D- arabino

hexopyranosyl]-D- chiro -inositol) in or on the commodity.

HED is now recommending the revocation of th e established tolerance in imported fruiting vegetables at 0.04 ppm, concomitant with the esta blishment of a domestic crop group tolerance in fruiting vegetables, group 8-10, at 0.15 ppm.

HED has reviewed the available toxicological and residue chemistry studies for kasugamycin, and is recommending that the requested uses be registered, and that pe rmanent tolerances be

established in/on the commodities in Table 2.3.3, at the specified recommended levels.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 8 of 60 TABLE 2.3.3 Tolerance Summary for Kasugamycin.‡ Commodity Proposed Tolerance (ppm) Recommended Tolerance (ppm) Correct Commodity Definition

Comments Vegetable, fruiting, Group 8 0.15 0.15 Vegetable, fruiting, group 8-10 Pome Fruit, Group 11 0.25 None Fruit, pome, group 11-10 Additional data are required for apples.*

Walnut 0.04 0.07 Apple None 0.08 Provided th e label is amended to replace

“Pome Fruit” with “Pear” and “All Pome Fruits Other than Pear”, and to specify a 30-day PHI for pear, and a 90-day PHI for all pome fruits other than pear. Azarole None 0.08 Loquat None 0.08 Crabapple None 0.08 Mayhaw None 0.08

Medlar None 0.08 Pear, Asian None 0.08 Quince None 0.08 Quince, Chinese None 0.08 Quince, Japanese None 0.08 Tejocote None 0.08

Pear None 0.30 * In lieu of establishing a crop group tolerance in pome fr uits, adequate field trial data are available to support individual tolerances in the various pome fruit commodities. ‡Tolerance levels as recommended within this doc ument are presented excluding antibiotic resistance impacts/concerns.

2.2.4 Revisions to Petitioned-for Tolerances

All recommended tolerances were determined vi a the OECD tolerance calculation procedures.

Other than the tolerance in frui ting vegetables, all recommended tolerances differed from those proposed by the petitioner, due to use of th e OECD tolerance calculation procedures.

Although a pome fruit crop group tolerance (fruit, pome, group 11-10) was requested, HED is recommending for the establishment of individual to lerances in the commodities included in the crop group, since the residue data for apple, one of the representative commodities of crop group 11-10 reflected a different use pattern than for other members of the crop group. Specifically, the pre-harvest interval (PHI) used in the apple field trials was significa ntly greater than the proposed label PHI (90 days inst ead of the proposed 30). If a pome fruit crop group tolerance is still desired, additional apple field trials reflecting the 30-day PHI are required.

2.3 Label Recommendations

2.3.1 Recommendations from Re sidue Chemistry Reviews

The product label for Kasumin 2L (EPA File Symbol 66330-UNU) should be amended as stipulated below and revised petition S ections B and F should be submitted.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 9 of 60 General use directions: The label should spec ify that adjuvants may not be added to spray mixtures for crops other thanpome fruits and walnuts.

Rotational crop statement: No rotational crop restrictions ar e specified on the proposed label.

The label should be revised to specify a 30-day plant-back inte rval (PBI) for all crops except those which are on the label as directly treated crops (which may be plan ted at any time).

Pome fruits: Since the submitted residue data for apple and pear, the representative commodities of pome fruits, group 11-10, are not adequate to fulfill data requirements for a crop group tolerance at this time, “Pome Fruit” should be re moved from the label and replaced with “Pear”

and “All Pome Fruits Other than Pear”.

PHI: For all pome fruits other th an pears, the product label sh ould be amended to specify a minimum PHI of 90 days. For pears, the pr oposed PHI of 30 days is appropriate.

3.0 Introduction

3.1 Chemical Identity

The chemical structure and nomenclature of kas ugamycin are presented in Table 3.1 (below).

TABLE 3.1 Kasugamycin Nomenclature.

Chemical Structure

[·HCl ·H 2 O]
Empirical Formula C 14 H 25 N 3 O 9 [C 14 H 25 N 3 O 9 •HCl•H 2 O]

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-( α -iminoglycino)- α -D-arabino-hexopyranoside

or [5-amino-2-methyl-6-(2,3,4,5,6-pentahydroxycyclohexyloxy)tetrahydropyran-3- yl]amino- α -iminoacetic acid

CAS Name (Kasugamycin) 3-O-[2-amino-4-[(car boxyiminomethyl)amino]-2,3,4,6-tetradeoxy- α -D-arabinohexopyranosyl]-D-chiro-inositol O

O OH OH

OH OH O H C H
3

N H NH

O H

O NH
2Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 10 of 60 TABLE 3.1 Kasugamycin Nomenclature.

CAS Registry Number 6980-18-3 [19408-46-9]

End-Use Product Kasumin 2L

Chemical Class Aminoglycoside antibiotic fungicide

Known Impurities of Concern None

3.2 Physical/Chemical Properties

Kasugamycin is an aminoglycoside antibiotic that ha s high stability and water solubility. It has a low vapor pressure (0.013 mPa). Its major routes of degradation in the environment are aqueous photolysis

, aerobic biodegradation

, and hydrolysis. Kasugamycin and its degradates are likely to be mobile in soils, but are not persistent. Based on the available information, there is no indication that kasugamcyin is likely to bioaccumulate. A detailed description of the physicochemical properties of kasugamycin is provided in Table C (see Appendix C).

3.3 Pesticide Use Pattern

A liquid (L) formulation end-use product (EP) co ntaining 2% kasugamycin (by weight) is being proposed by Arysta for use as a foliar broad cast spray on fruiting vegetables, pome fruits, and walnuts. The product will be marketed under th e trade name Kasumin 2L, and is formulated with kasugamycin hydrochloride hydrate at 2.3% (by weight), which is equivalent to 2% kasugamycin (0.1667 lb ai/gal), the free base. Table 3.3 (below) summarizes the proposed use
patterns.

The PPE requirements proposed on the Kasumin 2L Agricultural Antibiotic label state that applicators and other handlers must wear a long- sleeved shirt, long pants, socks plus shoes, and chemical-resistant gloves.Kasugamycin Human Health Risk Assessment DP Barcode D376608Page11of60TABLE 3.3 Summary of Proposed UseDirections for Kasugamycin.Application Timing;Type; andEquipmentEP1[EPA FileSymbol]Use Rate(lb ai/A)Maximum #of Uses PerSeasonMaximumSeasonal UseRate (lb ai/A)PHI2(Days)Use Directions and Limitations3Fruiting Vegetables (Group 8-10)Disease developmentconditions; foliardirected or broadcast;ground.0.1667 lb ai/gal L[66330-UNU]0.021 3 0.063 1 Minimum spray volume of 50 GPA4waterby ground. The addition of a wetting agentor spreader/sticker may enhance efficacy ofthe product. The minimum retreatmentinterval (RTI) is 7 days.Pome Fruits (Group 11-10)Disease developmentconditions (duringbloom); foliar directedor broadcast; ground.0.1667 lb ai/gal L[66330-UNU]0.083 4 0.333 30 Application after petal fall is prohibited. Donot use alternate tree-row applicationmethod. Spray volume of 100 GPA water byground. Reduced spray volumes may beutilized for small trees where completecoverage can be obtained with <100 GPA.The addition of a wetting agent orspreader/sticker may enhance efficacy of theproduct. The minimum RTI is 3 days.WalnutsDisease developmentconditions; foliardirected or broadcast;ground.0.1667 lb ai/gal L[66330-UNU]0.042-0.0834 0.333 100 Do not use alternate tree-row applicationmethod. Spray volume of 100 GPA water byground. Reduced spray volumes may beutilized for small trees where completecoverage can be obtained with <100 GPA.The addition of a wetting agent orspreader/sticker may enhance efficacy of theproduct. The minimum RTI is 14 days.1. EP = End-use Product.2. PHI = Pre-Harvest Interval.3. No rotational crop restrictions were specified.4. GPA = Gallons per Acre.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 12 of 60 3.4 Anticipated Exposure Pathways

RD has requested that HED perform an assessmen t of human health risk to support Arysta’s request for kasugamycin uses on fruiting vegetabl es, pome fruit and walnuts. Humans may be exposed to kasugamycin through consumpti on of food and drinking water containing kasugamycin residues. In addition, occupationa l handlers may be exposed via the dermal and inhalation routes when they mix /load and apply the pesticide to growing crops. Finally, workers re-entering treated areas to conduc t crop-related activities may be exposed via the dermal route.

The current risk assessment cons iders these potential exposure pa thways, in conjunction with the hazard database, and the endpoints and doses selected for risk assessment.

3.5 Consideration of Environmental Justice

Potential areas of environmental ju stice concerns, to the extent possi ble, were considered for this human health risk assessment, in accordance with US Executive Order 12898, Federal Actions to Address Environmental Justice in Minority Populations and Low-Income Populations ( http://www.epa.gov/compliance/resour ces/policies/ej/exec_order_12898.pdf ).

As a part of every pesticide risk assessment, the Office of Pesticide Programs (OPP) considers a large variety of consumer subgroups according to well-established procedures. In line with OPP policy, HED estimates risks to population subgroups from pesticide exposures that are based on patterns of that subgroup’s food and water c onsumption, and activities in and around the home that involve pesticide use in a residential setting. Extensive data on food consumption patterns are compiled by USDA under the Continuing Survey of Food Intakes by Individuals (CSFII), and are used in pesticide risk assessments for all registered food uses of a pesticide. These data are analyzed and categorized by subgroups base d on age, season of the year, ethnic group, and region of the country. Additionally, OPP is ab le to assess dietary exposure to smaller, specialized subgroups, and exposure assessmen ts are performed when conditions or circumstances warrant. Whenever appropriate , non-dietary exposures based on home use of pesticide products, and the associated risk estim ates for adult applicators, and for toddlers, youths, and adults entering or pl aying on treated areas post-application are evaluated. Further considerations are currently in development, as OPP has committed resources and expertise to the development of specialized software and mode ls that consider expo sure to bystanders and farm workers, as well as lifestyle and traditio nal dietary patterns among specific subgroups.

4.0 Hazard Characterization and Dose-Response Assessment

This hazard characterization upda tes the previous risk assessments for kasugamycin, conducted in 2005 (D301735; W.T. Drew; 17 August 2005) and 2009 (D356204; W.T. Drew; 29 April 2009). Additional toxicology data have been submitted since the most recent review (see below).Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 13 of 60 4.1 Toxicology Studies Available for Analysis

Studies considered for this a ssessment include acute lethality (oral, dermal and inhalation routes), primary eye and dermal irritation, dermal sensitization, subchronic oral toxicity (rat, mouse and dog), rat 21-day dermal toxicity, dog chronic toxicity, rat combined chronic toxicity/carcinogenicity, mouse car cinogenicity, developmental toxic ity (rat and rabbit), rat twogeneration reproductive toxicit y, genotoxicity, rat metabolis m, rat acute and subchronic neurotoxicity, and mouse immunotoxicity. A dditional data submitted since the previous

assessment were the acute and subchronic neur otoxicity studies, mouse immunotoxicity study, rat 21-day dermal toxicity study, bacter ial reverse gene mutation assay, and in vitro mammalian cell clastogenicity assay. The oral and dermal studies are of appropriate routes for potential exposure scenarios. Study executive summaries for the most recently submitted data and for updated DERs are provided in Appendix A.4; refe r to the previous risk assessments (2005 and 2009) for all other executive summaries.

A 28-day inhalation toxicity study in the ra t is not available; however, HED’s HASPOC concluded that an inhalation study is not requ ired for kasugamycin, based on the toxicological profile, low application rates, low vapor pressure, and lack of residential uses (TXR #0052461). Furthermore, MOEs for inhalation exposure, calculated using a c onservative NOAEL and endpoint from an oral study, were all ≥ 58,000. This approach is, theref ore, considered protective of potential effects from occupati onal inhalation exposure; a 10X UF DB is not required for the lack of this study.

4.2 Absorption, Distribution, Metab olism, & Elimination (ADME)

Kasugamycin was poorly absorbed and metabolized (<5% of the administered radioactive dose, or AD) in the rat following oral exposure to 100 or 1000 mg/kg/day. Absorption at low dosing levels (such as 1 or 10 mg/kg) was not examine d. The maximum plasma concentration occurred

about one hour after the administration of a single low or high dose, with a t 1/2 of 1.17-1.55 hours. Bioavailability, as indicated by the ar ea under the curve (AUC) was proportional to the dose. At 168 hours post-dosing, the majority of th e AD was excreted in the feces (81.9 to 93.9% AD), with only small amounts in th e urine (1.26-3.07% AD). Most of the excreted radioactivity was recovered within 48 hours. Biliary excret ion was not observed; the fecal radioactivity therefore represented unabsorb ed kasugamycin. By 168 hours, tissue radioactivity was nondetectable except for low levels in the kidneys, and at the high dose, the intestinal tract and stomach. Most of the excreted radioactivity in the urine and feces was the un-metabolized parent compound, and metabolism was not affected by dose, duration of dose, or sex. Minor amounts (less than 1% AD) of the metabolite kasuganobios amine were identified in the urine, liver, kidney and plasma, but none was detected in the feces.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 14 of 60 4.2.1 Dermal Absorption

A dermal absorption study is not available fo r kasugamycin. However, a dermal study was available to assess toxicity via the dermal route. No systemic effects were observed at doses causing dermal irritation at the application site. Females were more sensitive than males; the high dose of 500 mg/kg/day was reduced to 200 mg /kg/day in females after one week due to significant irritation.

4.3 Toxicological Effects

Kasugamycin is an aminoglycoside antibiotic pes ticide with limited activ ity against some plant bacterial and fungal pathogens. It does not have human or veterinary th erapeutic applications due to low efficacy, but at one time wa s used clinically in Japan to treat Pseudomonas kidney infections in humans (Shuwirth et al (2006) Nat. Struct. Mol. Biol. 13(10):879-886). The mode

of action is distinct from other aminoglycosides such as streptom ycin, which also has pesticidal uses. Kasugamycin inhibits the formation of th e 30S ribosomal subunit at initiation of protein synthesis by perturbing the mRNA-tRNA codon/ anticodon interaction. Other aminoglycoside

antibiotics bind to the 30S ribosom al subunit, but disrupt translati on of mRNA at later stages of

initiation.

Evaluation of metabolism in the rat indicates that, although peak plasma concentrations occur rapidly, absorption is poor. Kasugamycin is ra pidly excreted, largely as un-metabolized and unabsorbed parent compound, in the feces. Kasugamyci n is not retained at significant levels in

tissues. Metabolism is described in detail in Section 4.2 (above).

The primary target organs identified for kasuga mycin were the testes a nd kidney in the rat and mouse. However, these effects were only seen at higher dose levels, ge nerally at the highest dose tested (HDT). In the combined chronic toxi city/carcinogenicity stu dy in rats, the basis for

the LOAEL was an increased incidence and seve rity of testicular tubular atrophy, observed during the histopathologic examinati ons at the end of the two-year dosing period, as well as at 6 months, and one year. Testicular degeneration and atrophy were observed in adult F1 males in the rat reproductive toxicity st udy at the highest dose. Test icular tubular dilatation and degeneration were observed in the mouse subchroni c study, but at a dose that exceeded the limit dose; the mouse carcinogenicity study tested at much lower doses, and these effects were not observed. In the dog chronic t oxicity study, testicular inflam mation was reported at the high dose, but was not accompanied by atrophic or dege nerative changes, and was not considered a

treatment-related adverse effect.

Kidney toxicity is often associated with e xposure to aminoglycoside antibiotics, and the metabolism study indicated higher leve ls of radioactivity in the kidneys than other tissues. In male F1 rats in the reproductive toxicity study, dilatation of the ki dney, and an increased incidence of chronic progressive nephropathy we re observed. In the subchronic rat study, anKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 15 of 60 increased incidence of eosinophili c bodies (graded slight for seve rity) in the renal proximal tubular cells was reported in males at several dose levels. These eff ects were considered treatment-related, but not adverse due to their low se verity grade, and lack of associated findings. However, in female rats, increased epithelial ce lls in the urinary sedime nt, along with decreased urine pH (decreased pH was also seen in males) , were observed at the high dose, and considered evidence of possible kidney toxicity. Lipofusci n deposition (slight) was observed in the rat combined chronic toxicity/carcinog enicity study, but was not consid ered adverse due to the lack of other related findings; this study tested up to the NOAEL of the subchronic study. In the mouse, following subchronic exposure, minimal to severe basophilia/hyper plasia in the renal

pars recta in females was observed. No renal effect s were reported in the mouse carcinogenicity study at lower doses, or in the dog su bchronic or chroni c studies.

Kasugamycin caused decreased body weight and/or we ight gain in the subchronic studies in the rat, mouse and dog (including the neurotoxicity scr eening study in the rat). The chronic studies, which tested at lower doses, did not show body we ight effects. Decreased body weight was also observed in developmental and reproductive studies in the rat, and in the range-finding study for the rabbit developmental study. Body weight eff ects in the mouse immunotoxicity study were observed only at a dose significantly exceeding the limit dose.

Kasugamycin appears to be irritati ng to the oral and gastrointestin al tract mucosa. Anal lesions and perianal/perigenital staini ng were observed in the subchronic mouse study. Red and swollen skin around the anal opening, and inflammation and ul ceration of the rectum, were noted in rats of both sexes in both generations in the tw o-generation reproduction study. In the rat developmental toxicity study, distention of the la rge intestine with stool in the cecum, and an increased incidence of loose stool were reporte d. Similar findings we re seen in the rabbit developmental range-finding study among fema les that died or were sacrificed in extremis. These effects may be related to the acidity (or ot her irritant property) of the active ingredient, which is primarily excreted unabsorbed and un-meta bolized in the feces. Similar findings were not seen in the dog, although tongue and mouth lesions were reported at the highest dose tested in the dog subchronic toxicity study (but not the chronic dog st udy, which tested at a lower dose).

The available studies, which include acute and s ubchronic neurobehavioral screening studies in the rat, did not show evidence of neurotoxicity. A 28-day mous e immunotoxicity study did not show evidence of immune system effects.

Although kasugamycin did not show dermal irri tation following an acute exposure, it was irritating to the skin following re peated dermal dosing. Systemic effects were not observed in the rat 21-day dermal study, in which dosing wa s not conducted up to the limit dose, due to significant dermal irritation at the application site following repeated dose applications.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 16 of 60 There was no evidence of increased quantitative or qualitative susceptibility in rat or rabbit developmental toxicity studies, or in the rat re productive study. No developmental effects were seen in the rat developmental study, whereas ma ternal toxicity (decr eased body weight gain, food consumption, and feed efficiency) was obser ved at the highest dose. Although no maternal or developmental toxicity was observed in the ma in rabbit developmental toxicity study, in the dose range-finding study, maternal weight lo ss, reduced food consumption during dosing, and abortions (occurring at GD 18 or later) were ob served at higher doses. Fetal weight was decreased at the maternally toxic dose but, due to abortions or maternal death, was not evaluated at the higher doses. In the rat reproductive toxicity study, parent al toxicity included decreased parental body weight/weight gain at the mid and high doses. No of fspring toxicity was observed. Reproductive toxicity was observed only at the highest dose tested (above the parental LOAEL), with testicular atrophy, d ecreased fertility and fecundity in the F1 parents for both litters, and an increased pre-coital interval during th e mating period for the F2b litter.

Kasugamycin is classified as “not likely to be carcinogenic to humans ”, based on lack of evidence of carcinogenicity in rat and mouse car cinogenicity studies, and lack of evidence of genotoxic potential in gene mutation, chromoso mal aberration, and unscheduled DNA synthesis

assays.

The acute toxicity of kasugamycin is low by all rout es (Category III-IV). It is a mild dermal and ocular irritant (Category III), and does not show potential for dermal sensitization.

4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

HED recommends that the 10X FQPA safety factor for infants and children be reduced to 1X for kasugamycin, for the following reasons:

1. The toxicology database is complete , including rat acute and subchronic neurotoxicity screening studies , and a mouse immunotoxicity study.

2. Based on the lack of observed neurotoxicity, a DNT study is not required. 3. There is no evidence of increased qu antitative or qualitative pre- and/or postnatal susceptibility observed in developmental toxicity studies in the rat and rabbit, or in a two-generation reproduction study in

the rat. 4. The exposure assessment for fo od and drinking water will not underestimate potential dietary expo sure to kasugamycin, and there are no proposed or existing residential uses for kasugamycin.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 17 of 60 4.4.1 Completeness of the Toxicology Database

The database is considered adequate for purposes of assessing the suscepti bility of infants and children to the effects of kasugamycin. Acceptabl e developmental toxicity studies in two species (rat and rabbit), a rat multigen eration reproductive toxicity st udy, rat acute and subchronic neurotoxicity studies, and a mous e immunotoxicity study are availabl e. As previously discussed (Section 4.1), an inhalation e xposure study is not required.

4.4.2 Evidence of Neurotoxicity

Kasugamycin did not show evidence of neurotoxici ty in the available studies, which included rat acute and subchronic neurotoxicity studies. Ad ditionally, no potential neurotoxic effects were noted in other toxicity studies, or in ot her species. A DNT study is not required.

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal

There was no evidence of increased quantitative or qualitative pre- and/or postnatal susceptibility in developmental toxicity studies in two species, or the rat tw o-generation reproductive toxicity study. Abortions and a reduction in fetal body weight in the rabbit developmental toxicity rangefinding study were considered secondary to mate rnal toxicity (weight loss, and decreased food consumption). Decreased fertility and testicul ar effects in the F1 generation of the rat reproductive toxicity study were obs erved at the LOAEL for parental toxicity, but no toxicity to offspring was observed.

4.4.4 Residual Uncertainty in the Exposure Database

There is low concern, and no resi dual uncertainty, for susceptibil ity for the reasons discussed above. Underestimation of exposure is not an ticipated because conservative assumptions (tolerance-level crop residues, a nd modeled drinking water estimates) were used to estimate risk from dietary exposure, and there are no ex isting or proposed re sidential uses.

4.5 Toxicity Endpoint and Point of Departure Selections

4.5.1 Dose-Response Assessment

In the available studies, many of the effects observed following exposure to kasugamycin were only observed at the highest dose tested. Test icular atrophy was observe d at the highest dose levels in the mouse subchronic study, and in the rat chronic and reproducti ve toxicity studies. Decreased body weight and/or we ight gain was also usually obs erved only at the highest dose tested. Renal toxicity, observed in some studi es in the rat and mouse, was also a high-dose effect. Neither the chronic dog, nor the mouse carcinogenicity st udy, showed any effects at the doses tested, which were lower than thos e tested in the subchronic studies.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 18 of 60 For acute dietary exposure, a dos e and endpoint were not selected because there were no findings attributable to a single dose . Abortions in the rabbit ra nge-finding study at 30 and 100 mg/kg/day (1 of 4 and 3 of 4, respectively) we re only observed at or beyond 12 days of dosing

and in females that died or were sacrificed in extremis occurred after multiple doses. Maternal body weight loss during dosing and d ecreased food consumption were also observed at this dose, but are not considered a singl e-dose effect, and decreased fe tal weights were considered secondary to maternal toxicity.

For chronic dietary exposure, the rat combined chronic toxi city/carcinogenicity study was selected. The NOAEL of 11.3 mg/k g/day was based on testicular softening and atrophy at the LOAEL of 116 mg/kg/day. Testicular atrophy is consistent with findi ngs of other studies. It was

also observed in the rat tw o-generation reproduction study, at 425.3 mg/kg/day (NOAEL = 70.3 mg/kg/day) and in the mouse subchr onic study above the limit dose.

For dermal exposure (short- and intermediate-t erm), a dose and endpoint were not selected because no systemic toxicity was observed in the rat 21-day dermal study, which tested up to doses causing local dermal irritation. Endpoints of concern (body weight, renal and testicular toxicity, gastrointestinal and oral cavity effects) were eval uated in the dermal study.

For inhalation exposure (short- and intermediate -term), the subchronic study in the dog and the rabbit developmental toxicity stud ies were selected as co-critical . The endpoint of concern is irritation since kasugamycin appears to be irritati ng to the oral cavity and gastrointestinal tract mucosa following oral exposures. In the subchronic study with dogs, the NOAEL was 11 mg/kg/day, and the LOAEL was 106 mg/kg/day, based on irri tation of the oral cavity, characterized as tongue lesions, swollen mouth, ex cessive salivation, and thickening of the skin at the commissures of the mouth. The irritation po tential of kasugamycin is further demonstrated with the presence of anal lesions, and perianal/p erigenital staining seen in the subchronic mouse study; red/swollen skin around the anal opening, and inflammation/ ulceration of the rectum in the offspring of both sexes in the two-generatio n reproduction study; and distention of the large intestine in the developmental rat study. The deve lopmental rabbit study was selected as the cocritical study since the matern al NOAEL (10 mg/kg/day) was co mparable to the NOAEL in the dog study, and the concern for the maternal toxicity seen in this species. In the range-finding study, maternal toxicity manifested as abortions and weight loss at 30 mg/kg/day (LOAEL). In the rabbit developmental toxicity studies (range-finder and definitive evaluated together), the incidence and severity of the ma ternal toxicity was increased at the high dose (100 mg/kg/day) where one doe died, abortions were seen in 3 of 4 litters, and the weight loss was more pronounced. Given kasugamycin’s apparent poor or al absorption (< 5% in the rat metabolism study), and the consistent evidence of irritati on effects to mucous membranes seen across numerous species, it is reasonable to surmise that the bolus (gavage) dosin g causes irritation to the gastrointestinal tract resulting in maternal toxicity. Additionally, the use of the oral NOAELKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 19 of 60 results in MOEs ranging from 58,000 to 2,500,000 for risk from occupational exposure via the

inhalation route.

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

For chronic dietary assessment, exposure from food and drinking water should be combined. There are no residential uses at this time, so it is not necessary to address the suitability of combining exposures from differe nt routes (such as oral and inhalation). Only inhalation exposure was assessed for occupational workers, since there was no haza rd associated with dermal exposure.

4.5.3 Cancer Classification and Ri sk Assessment Recommendation

In accordance with the Agency’s 2005 Guid elines for Carcinogen Risk Assessment, kasugamycin is classified as “not likely to be carcinogenic to humans ”, based on lack of evidence of carcinogenicity in male and female mi ce, and in male and female rats, and the lack of evidence for mutagenicity both in both in vivo and in vitro assays.

4.5.4 Summary of Points of Departure and T oxicity Endpoints Used in Human Risk

Assessment

TABLE 4.5.4.1 Summary of Kasugamycin Toxicologi cal Doses and Endpoints for Use in Dietary

Human Health Risk Assessments. Exposure Scenario Point of Departure Uncertainty and FQPA Safety Factors RfD, PAD, LOC

for Risk

Assessment Study and Toxicological Effects

Acute dietary (all populations) An appropriate dose and endpoint for this risk assessment scenario was not identified, based on a lack of single-dose effects in the database. Chronic dietary (all populations including infants and children, and females age 13 to 49) NOAEL = 11 mg/kg/day UF A = 10X

UF H = 10X FQPA SF = 1X Chronic RfD = 0.11 mg/kg/day

cPAD = 0.11 mg/kg/day Combined chronic toxicity/carcinogenicity study in the

rat

LOAEL = 116 mg/kg/day, based on testicular atrophy and softening. Cancer (oral, dermal, inhalation) Classification: “Not likely to be carcinogenic to humans.”

Point of Departure (POD) = a data point or estimated point derived from observed dose-response data, which is used to mark the beginning of extrapolation to determine risk associated with lower envi ronmentally relevant human exposures. HDT = Highest Dose Tested. UF = Uncertainty Factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. NOAEL = No Observed Adverse Effect Level. LOAEL = Lowest Observed Adverse Effect Level. PAD = Population Adjusted Dose (a = acute, c = chronic). RfD = Reference Dose. LOC =

Level of Concern.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 20 of 60 TABLE 4.5.4.2 Summary of Kasugamycin Toxicolo gical Doses and Endpoints for Use in Occupational Human Health Risk Assessments. Exposure

Scenario Point of Departure Uncertainty

Factors RfD, PAD,

LOC for Risk

Assessment Study and Toxicological Effects

Dermal, shortterm (1 to 30 days) and

intermediateterm (1 to 6 months) An appropriate dose and endpoint for these scenarios was not identified. No toxicity was observed in the rat 21-day dermal toxicity study, which tested up to doses causing local dermal

irritation.

Dermal, longterm (>6 months) Long-term dermal exposure is not anticipated from the proposed or existing uses.

Inhalation, short-term (1 to 30 days) and

intermediateterm (1 to 6 months) Oral NOAEL = 11 mg/kg/day* UF A =10X

UF H =10X Occupational

LOC for MOE

≤ 100 Co-critical studies:

1. Subchronic oral toxicity in the dog; LOAEL = 106 mg/kg/day

2. Developmental toxicity study in rabbits. Maternal LOAEL = 30 mg/kg/day

Endpoint of concern is irritation (see Section 4.5.1 for details).Inhalation longterm (>6 months) Long-term inhalation exposure is not anticipat ed from the proposed or existing uses.

Cancer (oral, dermal, inhalation) Classification: “Not likely to be carcinogenic to humans.”

Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data, which is used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. UF = Uncertainty Factor. UF A = extrapolation from animal to human (interspecies).

UF H = potential variation in sensitivity among members of the human population (intraspecies). NOAEL = No

Observed Adverse Effect Level. LOAEL = Lowest Observed Adverse Effect Level. MOE = Margin of Exposure.

LOC = Level of Concern. * Absorption via the inhalation route is equivalent to 100% oral absorption.

5.0 Dietary Exposure/Risk Characterization

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

The nature of kasugamycin residues in plants and rotational crops is considered adequately understood for the purpose of assessing the pr oposed uses, based on an acceptable tomato metabolism study and an acceptable confined rota tional crop study. The residue of concern inKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 21 of 60 fruiting vegetables for both tolerance enforcem ent and risk assessment purposes is the parent kasugamycin only, and this conclusion applies to both walnuts and pome fruit. HED concluded that a metabolism study is not required for walnut s, since the maximum field trial residues were at the limit of quantitation (LOQ), 0.040 ppm, and are therefore unlikely to be present in quantities sufficient to allow char acterization. For any future requested uses on leafy, root or grain crops (or other crops dissimilar to tree nuts, fruiting vegetables, or pome fruits), metabolism studies would be require d in order to determine the nature of the residue in those commodities. No rotational crop tolerances are n eeded, based on the overall lack of residues in rotated crops.

Based on the results of the goat metabolism st udy, kasugamycin residues orally ingested by a lactating goat were poorly absorbed from the ga stro-intestinal (GI) trac t of the goat, and the small fraction of the dose that was absorbed was rapidly excreted unchanged in the urine. Kasugamycin did not accumulate in the milk or ed ible tissues of the goat. The nature of the residue is adequately unders tood, and HED has recommended use of the parent compound only for purposes of tolerance enforcement and risk assessment. However, based on the proposed uses, there is no reasonable expectation of fin ite residues in livestock commodities (a category 6[a][3] situation), so no tolerances are being recommended in livestock commodities at this time.

5.1.2 Summary of Environmental Degradation

EFED provided a fate summary and drinking wa ter assessment (DWA) for the proposed uses (D376600; T. Dowen, J. Hetrick, Dana Spatz; 28 Ap ril 2011). The primary routes of dissipation for kasugamycin are aqueous photolysis

, aerobic biodegradation

, and hydrolysis, w ith half-lives of 17.4, 45, and 11.4 to 462 days (for pH 4 to 9), respectively. The aerobic soil metabolism halflife was 73 days in clay-loam soil. Since aer obic soil metabolism data are only available on one soil type, there is uncertainty regarding the variab ility of degradation in aerobic soils. The field dissipation half-lives ranged from 5.7 to 12.3 da ys. The degradate kasugamycinic acid was not detected above the LOQ at any sampling interval. No other degradates were looked for in this study. Based on the FAO Mobility classification, kas ugamycin is classified as highly mobile (in sand) to moderately mobile (in clay loam soil). The mobility of kasugamycin acid and kasuganobiosamine is expected to be similar to, or greater than, the parent. Therefore, they will likely range from highly mobile to moderately mob ile. All have the poten tial to be found in both surface water and groundwater sour ces of drinking water.

5.1.3 Comparison of Metabolic Pathways

In a rat metabolism study, kasugamycin was poor ly absorbed following oral exposure, and excreted almost entirely in the feces as un-meta bolized parent compound. Excretion in the urine

accounted for ≤ 3% of the dose. The metabolite kasuganobiosamine was identified at low levels in urine, liver, kidney and plasma (<1% of dose). Little or no radioactivity was retained in

tissues.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 22 of 60 In the tomato metabolism study, the metabolite prof ile was similar for tomato fruit and foliage.

Based on the results of the tomato metabol ism study, the major metabolic pathway of kasugamycin in plants involves conjugati on of the parent compound, conversion to kasugamycinic acid, and subs equent conjugation of kasuga mycinic 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.

The submitted data shows that the major metabolic pathway of kasugamycin in tomato fruit and

leaves consists of deamination and oxidat ion to produce kasugamycinic acid. Metabolism studies in rotational crops (lettu ce, radish and wheat) show uptake of kasugamycin in all crops, while kasugamycinic acid was identified in onl y one commodity, wheat hay. The livestock metabolism study conducted in goat showed kas ugamycin as the only residue identified in the liver and kidney. Similarly, parent compound wa s the major residue iden tified in the urine, feces, liver, kidney and plasma of rats.

Compounds detected in the tomato, ruminant, ro tational crop, and rat metabolism studies are presented in Tables B.1 a nd B.2 (see Appendix B).

5.1.4 Residues of Concern Summary and Rationale

Kasugamycin is susceptible to aqueous photolys is, aerobic biodegradation and hydrolysis. It is highly soluble in water, and doe s not appear to volatilize sign ificantly. Adsorption-desorption studies indicate that kasugamyci n is highly to moderately mobile in soil. Major identified degradates of kasugamycin include kasugamy cinic acid and kasuganobiosamine. The soil mobility of these two metabolites is expected to be similar to, or greater than, the parent kasugamycin. Therefore, both of these degradates have the potenti al to be found in surface and ground sources of drinking water.

Although kasugamycinic acid, 2-N-acetyl-kasuga mycin and/or kasuganobiosamine were detected in primary crops, rotational crops, and/or water, these metabolites have similar structures to the parent, and thus can be reasonably expected to have similar toxicity. Moreover, a DEREK analysis was conducted for these three me tabolites, and major differences in toxicity were not predicted rela tive to the parent.

Water: The ROCKS committee recomm ended that parent kasugamycin, as well as its major degradation products kasugamyci nic acid and kasuganobiosamine, be included in the risk

assessment.

Plants: Kasugamycin accounts for 55-94% of the to tal radioactive residu e (TRR) in tomato fruit, and 52-84% of the TRR in tomato leaves fo r PHIs from 2 hours to 28-days. Therefore, theKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 23 of 60 parent compound can serve as an adequate meas ure of misuse for tolerance enforcement. Kasugamycinic acid accounted for 10% and 12% of the TRR in tomato fruit at 21-day and 28- day PHIs, respectively, compared to only 2-7% of the TRR in tomato leaves at all PHIs. Kasugamycinic acid seems to be formed at l ong PHIs; the proposed PHI is 1-day for fruiting vegetables. In addition, crop fi eld trials with walnut show kasugamycin residues below the LOQ. Although the PHIs for pears (30 days) an d apples (90 days) will be longer than the 28 days for tomatoes in the metabolism study, ROCKS c oncluded that use of only the parent in risk assessment is adequate for the proposed uses, based on the relatively low toxicity, and low residues of the parent. Moreover, no special concerns regarding the potential toxicity of kasugamycinic acid were noted. Based on this information, the ROCKS recommends that parent kasugamycin only be used for tolerance enforcement and risk assessment. However, for further petitions of new uses on fruiting-type crops w ith longer PHIs, the crop field trial data should include analysis for kasugamycinic acid. For futu re uses on other types of crops (such as leafy vegetables, or grains) a new metabolism study will be required which should quantitatively assess the formation of kasugamycinic acid.

Rotational Crops: In the limited field rotational crop trials with lettuce, radish and wheat planted at 30, 120 and 365 days after soil treatment, uptak e of kasugamycin was observed at levels below 0.01 ppm. The only metabolite identified was ka sugamycinic acid in wheat hay, but only at levels below 0.001 ppm. Based on this, th e ROCKS committee reco mmended that parent kasugamycin only be included for risk assessment and tolerance enforcement purposes.

Livestock: The only residue identified in a goat metabolism study was kasugamycin, accounting for 92.9% TRR in the liver and kidney, and 85.7% TRR in renal fat. Kasugamycin is not metabolized to a major extent. Based on this , the ROCKS committee re commended that parent kasugamycin only be included for risk assessment and tolerance enforcement purposes.

TABLE 5.1.4 Summary of Metabolites and Degradates to be Included in Ri sk Assessments and the Tolerance Expression.

Matrix Residues Included in Risk

Assessments Residues Included in the Tolerance Expression Plants Primary crop Parent kasugamycin only Parent kasugamycin only Rotational crop Parent kasugamycin only Parent kasugamycin only Livestock Ruminant Parent kasugamycin only Parent kasugamycin only Poultry NA* NA Drinking Water Kasugamycin, kasugamycinic acid, and kasuganobiosamine NA

* NA = Not Applicable.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 24 of 60 5.2 Food Residue Profile

Analytical chemistry and residue data for kasuga mycin were addressed in detail in the summary document associated with the current peti tion (D376563; W.T. Drew; 13 July 2012). All

recommended tolerances were determined via the OECD tolerance calcu lation procedure.

The submitted and available residue data for fruiting vegetables, group 8-10, and walnuts are adequate to fulfill data requirements. For pome fru it, the data are adequate to support individual crop tolerances, but not the crop gr oup tolerance. If the petitioner wishes to pursue a crop group tolerance in pome fruits, group 11-10, residue data from 6 additional geographically diverse apple field trials (including adjuvant) are requi red at a 30-day PHI. The submitted data are adequate to support individual tolerances in pome fruit commodities, provided the label is amended to specify a 90-day PHI for all pome fruit crops other than pears.

Adjuvants were not used in any of the fruiting vegetable or walnut field trials. However, in the case of walnuts, HED concludes that adjuvant s will not have a significant impact on residues, given the long PHI, the low observed residues in the available trials, and the nutmeats being protected (by the walnut’s shell) from direct spray during application. Therefore, HED is recommending that the product label be amended to specify that adjuvants may not be added to spray mixtures for crops other than pome fruits and walnuts.

Adequate processing studies were conducted with apples and tomatoes. The processing study in apples demonstrated that residue s concentrated slightly (1.5X) in apple juice, but a separate tolerance for residues in juice is not needed. No processing factor coul d be calculated for wet pomace because residues were below the LOQ in pomace. No processing factors could be calculated for tomato processed commodities because there were no detectable residues in the RAC or the processed fractions. The Agency woul d have preferred the us e of exaggerated rates in the conduct of the processing study to yield a RAC with quantifiable residues. However, in conjunction with the previous petition (PP#3E6579; D308573; W.T. Drew; 17 August 2005), the petitioner provided supporting information, and wa s granted a waiver from the requirement for another tomato processing study. Si nce the proposed use is nearly identical to the previous use directions for fruiting vegetables, HED con tinues to recommend for a waiver from the requirement for a new tomato proce ssing study at exaggerated rates.

A summary of the crop fiel d trial residue data is presented in Table 5.2.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 25 of 60 TABLE 5.2 Summary of Residue Data from Crop Field Trials with Kasugamycin.

Commodity Total Use Rate (lb ai/A) PHI (Days) Residue Levels (ppm) 1

n Min. Max. HAFT Median Mean Std.

Dev.
Fruiting Vegetables (proposed use = 0.063 lb a i/A total use rate, 1-day PHI, plus adjuvant) Data submitted in current petition (PP#0F7689) Pepper fruit (foliar broadcast) 0.0619-0.0714 1 18 <0.04 0.107 0.0837 0.0400 0.0472 0.0167

Tomato fruit (foliar

directed/foliar

broadcast) 0.0623-0.0687 0-1 20 <0.04 0.0728 0.0727 0.04 0.0441 0.0105

Tomato fruit

(drench) 0.0624 lb ai/ 25 gal water 0 2 <0.04 <0.04 <0.04 <0.04 <0.04 NA 2

Data available from the previous petition (PP#3E6579) 3
Pepper fruit 0.053-0.054 1 14 <0.04 <0.04 <0.04 <0.04 <0.04 NA

Tomato fruit 0.052-0.054 1 16 <0.04 <0.04 <0.04 <0.04 <0.04 NA

0.270 1 2 0.044 0.056 0.050 NR 4 0.050 0.008
Pome Fruit (proposed use = 0.333 lb ai/A total application rate, 30-day PHI, plus adjuvant) Data submitted in current petition (PP#0F7689) Kasumin 2L + Surfactant (All Trials) Apple fruit 0.261-0.341 90-100 40 <0.04 0.075 0.068 0.04 0.043 0.007 Kasumin 2L + Surfactant (Canadian Side-by-SideTrials) Apple fruit 0.325-0.341 91-94 10 <0.04 <0.04 <0.04 <0.04 <0.04 NA Kasumin 2L Only (Canadian Side-by-Side Trials) Apple fruit 0.325-0.341 91-94 10 <0.04 <0.04 <0.04 <0.04 <0.04 NA Kasumin 2L + Surfactant (All Trials)

Pear fruit 0.494-0.538 27-32 16 0.044 0.172 0.166 0.100 0.097 0.038 Data associated with Section 18 emergenc y exemption for use on apples (PP#08MI10) 3
Apple fruit 0.32 30 6 0.014 0.029 0.025 0.021 0.022 0.006
Walnuts (proposed use = 0.333 lb ai/A total application rate, 100-day PHI, plus adjuvant)

Walnut nutmeat 0.335-0.340 98-110 6 <0.04 0.04 0.04 0.04 0.04 0
1. The LOQ is 0.040 ppm. 2. NA = Not Applicable. 3. D308573; W.T. Drew; 17 August 2005; PP#3E6579. 4. NR = Not Reported. 5. D356204; W.T. Drew; 29 April 2009; PP#08MI10.

5.3 Water Residue Profile

The kasugamycin drinking water re sidues used in risk assessment were provided in the DWA by EFED (D376600; T. Dowen, J. Hetrick, Dana Spat z; 28 April 2011), and in corporated directly into the dietary exposure and risk assessment. Water residues we re incorporated in the dietary assessment model using the food cat egories “water, direct, all sour ces” and “water, indirect, all

sources”.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 26 of 60 In general, estimated concentrations in drinki ng water sources are considered to be low, but surface water estimates were slightly highe r than those for groundwater. Although EFED provided values for acute, chronic and cancer as sessments, only the chronic surface water value was used quantitatively in the risk assessmen t. The EDWCs in surface water are 0.011562 ppm and 0.001178 ppm for acute and chroni c scenarios, respectively. The EDWC in groundwater is 0.000116 ppm for all durations (see Table 5.3.1). The chronic surface water EDWC (0.001178 ppm) was used in the chronic diet ary analysis because it is the higher of the chronic residues in water; use of this value in the dietary assessmen t is considered protectiv e of potential exposure to residues in ground water sources of drinking water.

The modeled surface water EDWCs were generate d using the linked PRZM (Pesticide Root Zone Model) and EXAMS (EXposure Analysis Modeling System) models. These models and their descriptions are available at th e EPA internet website via the web link, http://www.epa.gov/oppefed1/models/water/ . No surface or groundwater monitoring data were available for kasugamycin. The PRZM/EXAM S modeling was based on application of kasugamycin to tomatoes in Florida.

TABLE 5.3.1 EDWCs for Kasugamycin, Plus Degradates Kasugamycinic Acid and Kasuganobiosamine.

Crop Acute Exposure (ppm) Chronic Exposure (ppm) Cancer Exposure (ppm)

Surface Water (via PRZM/EXAMS)

Tomato 0.011562 0.001178* 0.000602

Ground Water (via SCI-GROW)

Walnut 0.000116 0.000116 0.000116

* EDWC used in the chronic dietary assessment.

5.4 Dietary Risk Assessment

5.4.1 Description of Residue Data and Percent Cr op Treated Used in Dietary Assessment

An unrefined chronic aggregate dietary (food and drinking water) exposure and risk assessment was conducted for kasugamycin (D390458; W.T. Drew; 13 July 2012) using the Dietary Exposure Evaluation Model software with th e Food Commodity Intake Database (DEEMFCIDTM), Version 2.03. This model uses food cons umption data from the US Department of Agriculture’s (USDA’s) Continuing Surveys of Food Intakes by Individuals (CSFII), collected from 1994 to 1996, and 1998. The residue inputs into the dietary model were the recommended tolerance level residues; defau lt processing factors were used, with the exception of the apple juice processing factor, for which the 1.5X data-derived factor was used. The dietary exposure assessment is based on the assumption of 100%CT for all crops with proposed uses on the kasugamycin label.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 27 of 60 5.4.2 Chronic Dietar y Risk Assessment

Based on these highly conservative assumptions, ch ronic dietary risk estimates are less than or equal to 1.7% of the cPAD for all population subgr oups. Children 1-2 years of age are the most highly-exposed subgroup, with a risk estimate of 1.7% of the cPAD, while the general US population had a risk estimate of <1% of the cP AD. Generally, HED is concerned when risk estimates exceed 100% of the PAD; therefore, all chronic dietar y risk estimates are not of concern (see Table 5.4.3).

5.4.3 Summary Table

TABLE 5.4.3 Summary of Chronic Dietary Exposure and Risk Estimates for Kasugamycin. Population Subgroup* [Years of Age] cPAD (mg/kg/day) Dietary Exposure Estimate (mg/kg/day) % cPAD

General US Population 0.11 0.000450 <1

All Infants [< 1] 0.11 0.001215 1.1

Children [1-2] 0.11 0.001839 1.7

Children [3-5] 0.11 0.001281 1.2

Children [6-12] 0.11 0.000647 <1

Youth [13-19] 0.11 0.000373 <1

Adults [20-49] 0.11 0.000310 <1

Adults [50+] 0.11 0.000279 <1

Females [13-49] 0.11 0.000307 <1

* Values for the population subgroup with the highest risk are in bold type.

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are no proposed residential uses for kasuga mycin associated with the subject petition, nor are there any currently re gistered residential uses. Kasugamy cin is not intended for use in public or residential settings. Therefore, residential ex posure is not expected, and residential risks are

not of concern.

6.1 Spray Drift

Spray drift is always a potential source of expos ure to residents living in close proximity to spraying operations. This situati on is particularly the case with ae rial application. However, to a lesser extent, spray drift resulti ng from the ground application of kasugamycin could also be a potential source of exposure. The Agency has b een working with the Spray Drift Task Force (a membership of US pesticide registrants), EPA Re gional Offices, various state lead agencies for pesticide regulation, and other part ies to develop the best spray dr ift management practices. On a chemical-by-chemical basis, the Agency is now requiring interim mitigation measures for aerial applications that must be placed on product labels. The Agency has completed itsKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 28 of 60 evaluation of the new database submitted by the Spray Drift Task Force, and is developing a policy on how to apply appropr iately the data and the AgDRIFT computer model to its risk assessments for pesticides applied by air, orch ard airblast, and ground hydr aulic methods. After the policy is in place, the Agency may impose further refinements in spray drift management practices to reduce off-target drif t and risks associated with aerial (as well as other) pesticide application methods, wh ere appropriate.

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate pesticide exposures and risk estimates from three major sources: Food, drinki ng water, and residential exposures. In an aggregate assessment, exposures from dietary and residential sources are added together. Since

there are no residential sources of exposure for kasugamycin, the onl y aggregate risk scenario is chronic dietary (food and water), which is not of concern (see Table 5.4.3).

8.0 Cumulative Exposure/Risk Characterization

Unlike other pesticides for which EPA has fo llowed 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 ka sugamycin does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has not assumed that kasugamycin has a common mechanism of to xicity with other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity, and to evaluate the cumulative effect s of such chemicals, see the policy statements released by EPA’s OPP con cerning common mechanism determinations and procedures for cumulating effects from subs tances found to have a common mechanism on

EPA’s website at http://www.epa.gov/pesticides/cumulative/

.

9.0 Occupational Exposure/Risk Characterization

Based on the proposed use patter ns, both short- and intermed iate-term exposure and risk assessments were conducted for occupational ha ndlers, or those who mix, load and apply pesticides. In addition, HED c onsidered the potential for post-application exposure for those reentering treated sites (D376591; S.C. Wang and C. Swartz; 26 June 2012).

9.1 Short- and Intermediate-Term Handler Exposure and Risk

No chemical-specific data were available to asse ss potential exposure of pesticide handlers to kasugamycin. In the absence of chemical-specific data, it is HED policy to use the best available data to assess handler exposure. Sources of generic handler data, used as surrogate data in the absence of chemical-specific data, include the Pesticide Handlers Expos ure Database Version 1.1 (PHED 1.1), and the Agricultural Handler Exposur e Task Force (AHETF) database. Some ofKasugamycin Human Health Risk Assessment DP Barcode D376608

Page 29 of 60 these data are proprietary (such as the AHETF data), and subject to the data protection provisions

of FIFRA. The standard valu es recommended for use in pred icting handler exposure that are used in this assessment, known as “unit exposures ”, are outlined in the Occupational Pesticide Handler Unit Exposure Surrogate Reference Table ( http://www.epa.gov/opp00001/scien ce/handler-exposure-table.pdf ), which, along with additional information on HED policy for use of su rrogate data, including descriptions of the various sources, can be found at: http://www.epa.gov/pesticides/s cience/handler-exposuredata.html . The average adult body weight (80 kg) wa s used because the dose and endpoint

selected for inhalation risk a ssessment are not sex-specific. The proposed label was used to determine the maximum application rate.

There are 5 handler scenar ios that are expected to result in the highest exposure for the proposed

uses:

1. Mixing/loading liquids for air-blast application, 2. mixing/loading liquids fo r ground-boom application, 3. applying sprays using air-blast equipment, 4. applying sprays using gr ound-boom equipment, and 5. mixing/loading/applying sprays using a manually pressurized

handwand.

No data on the number of exposure days per year was provided. For this risk assessment, HED assumed that most handlers would be exposed for less than 6 months (short-term and intermediate-term exposure). However, since th e short- and intermedia te-term toxicological endpoints are the same, only short-term exposure was quantitatively assessed, and is considered protective of potential intermediate-term exposure.

Based on HED’s Exposure Science Advisory Council (ExpoSAC) Policy #9.1, the following assumptions were used for acres treated per day:

1. 40 acres/day for air blast applications, 2. 80 acres/day for ground-boom applications, and 3. 0.8 acres/day (assuming 40 gal/day for use of a manually pressurized handwand); the draft label propos es using a spray volume of 50 GPA for fruiting vegetables.

The handler exposure estimates in this assessmen t are based on a central tendency estimate of unit exposure, and an upper-percentile assumption for the application rate. The uncertainties

associated with this assessment stem from the us e of surrogate exposure data (such as differences in use scenario, and data confidence), and a ssumptions regarding the amount of chemical handled. The estimated exposures are believed to be reasonable high -end estimates, based on observations from field studies, and professional judgment.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 30 of 60 All MOEs for handlers are greater than 100 (ranging from 58,000 to 2,500,000), and are not of concern. The exposure and risk estimates for occupational handlers using kasugamycin are presented in Table 9.1.Kasugamycin Human Health Risk Assessment DP Barcode D376608Page31of60TABLE 9.1 Non-Cancer OccupationalHandlers’ Exposure and RiskEstimates for Kasugamycin.Exposure Scenario[Scenario Number, DataSource]MitigationLevel1Inhalation UnitExposure(μg/lb ai)CropsApplication Rate(lb ai/A or lbai/gal)Amount Treated2(A/day or galused/day)DailyInhalationDose3(mg/kg/day)InhalationMOE4Mixer/LoaderLiquids for Air BlastApplication [1, AHETF]Baseline 0.219 Pome FruitWalnuts0.08 40 0.00000876 1,300,000Liquids for Ground-BoomApplication [2, AHETF]Baseline 0.219 FruitingVegetables0.02 80 0.00000438 2,500,000ApplicatorSprays with Air BlastEquipment [3, AHETF]Baseline 4.71 Pome FruitWalnuts0.08 40 0.00018840 58,000Sprays with GroundBoom Equipment [4,AHETF]Baseline 0.34 FruitingVegetables0.02 80 0.00000680 1,600,000Mixer/Loader/ApplicatorSprays with ManuallyPressurized Handwand [5,PHED]Baseline 30 FruitingVegetables0.0004 lb ai/gal40 gal 0.000006 1,800,0001. Baseline Inhalation Exposure represents no respiratory protection, open mixing/loading, and open cab tractors, as appropriate.2. Daily acres treated values are from EPAestimates of acreage that could be treatedor volume handled in a single day for each exposure scenario of concern,based on the application method and formulation/packaging type.3. Daily Inhalation Dose (mg/kg/day) = [unit exposure (μg/lb ai)* (1mg/1000 μg) conversion * application rate (lb ai/acre or lb ai/gal) * daily acres treated or galused /day] / body weight (80 kg).4. Inhalation MOE = NOAEL (11 mg/kg/day) / Daily inhalation dose (mg/kg/day).Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 32 of 60 9.2 Short- and Intermediate-Term Po st-Application Exposure and Risk

9.2.1 Dermal Post-Application Risk

Dermal post-application exposure was not assessed due to the lack of toxicity via the dermal

route. There is no dermal risk associated with post-application activities following application of kasugamcyin on the proposed crops. The Restricted Entry Interval (REI) is based on the acute toxicity of kasugamycin technical material, which is classified in acute Toxicity Categories III and IV. Acute Category III or IV chemicals require a 12-hour REI. Thus, the 12-hour REI that appears on the labels is adequate.

9.2.2 Inhalation Post-Application Risk

Based on the Agency's current practices, a quant itative post-application inhalation exposure assessment was not performed for kasugamycin at th is time, primarily because it has a low vapor pressure (vapor pressure <0.013 mpa at 25oC), and it is applied at low ap plication rates. However, volatilization of pestic ides may be a potential source of post-application inhalation exposure to individuals nearby to pesticide applications. The Agency sought expert advice and input on issues related to volatilization of pe sticides from its Federal In secticide, Fungicide, and

Rodenticide Act Scientific Advi sory Panel (SAP) in December 2009. The Agency received the SAP’s final report on 2 March 2010 ( http://www.epa.gov/scipoly/SA P/meetings/2009/120109meeting.html ). Although a quantitative occupational post-application i nhalation exposure assessment was not performed, an inhalation exposure assessment was performed for occupational handlers. This assessment resulted in risk

estimates that did not exceed HED’s LOC at baseline inhalation PPE. Handler exposure resulting from application of pe sticides outdoors is likely to re sult in higher exposure than postapplication exposure. Therefore, it is expected that these handler inha lation exposure estimates would be protective of most occupational post-a pplication inhalation expo sure scenarios.

10.0 References

Risk Assessment Memorandum Kasugamycin. Human Health Risk Assessment for Proposed Food Uses of the Fungicide Kasugamycin on Imported Fruiting Vegetables (Group 8). ; D301735; W.T. Drew; 17 August

2005.

Residue Chemistry Summary Document Memorandum Kasugamycin. Petition to Establish Permanent Tolerances for Residues of the Fungicide Resulting from Food Use on Fruiting Vegetabl es, Pome Fruit, and Walnuts (PP#0F7689). Summary of Analytical Chemistry and Residue Data. ; D376563; W.T. Drew; 13 July 2012.Kasugamycin Human Health Risk Assessment DP Barcode D376608

Page 33 of 60 Occupational and Residential Expo sure Assessment Memorandum Kasugamycin: Occupational and Residential Exposure/Risk Assessment for Section 3 Registration on Pome Fruit, Walnuts, and on Fruiting Vegetables ; D376591; S.C. Wang; 26

June 2012.

Dietary Exposure Memorandum Kasugamycin. Chronic Aggregate Dietary (F ood and Drinking Water) Exposure and Risk Assessment for the Section 3 Registration Acti on Associated with Food/Feed Use of the Fungicide on Fruiting Vegetables, Pome Fruit, and Walnuts (PP#0F7689). ; D390458; W.T. Drew; 13 July 2012.

Drinking Water Assessment Memorandum Drinking Water Assessment for Tolerance Peti tion for New Chemical Kasugamycin [EPA Est No. 075703-JPN-001] ; D376600; T. Dowen, J. Hetrick, Dana Spatz; 28 April 2011.

ROCKS Decision Memorandum Kasugamycin. Report of the Residues of Concern Knowledgebase Subcommittee (ROCKS). ; D387527; I. Negrón-Encarnación; 4 May 2011.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 34 of 60 APPENDIX A: Toxicology Profil e and Executive Summaries

A.1 Toxicology Data Requirements

The requirements (40CFR §158.340) for food use of kasugamycin are shown in Table A.1, below. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

TABLE A.1 Toxicology Data Requirement s for Kasugamycin, Technical.

Test Required Satisfied

870.1100 Acute Or al Toxi city.....................................................

870.1200 Acute Derm al Toxicity ................................................

870.1300 Acute Inhala tion Toxic ity ........................................... 870.2400 Primary Ey e Irritation ................................................. 870.2500 Primary Derm al Irrita tion ............................................

870.2600 Dermal Sensitiza tion ................................................... yes

yes

yes

yes

yes

yes yes 1

yes 1

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 28-Day Inhalation........................................................ yes

yes

yes

no

no 2 yes

yes

yes

--

--
870.3700a Developmental Toxicity (rodent) ................................ 870.3700b Developmental To xicity (non rodent) .......................... 870.3800 Repr oduction .............................................................. yes

yes

yes yes

yes

yes

870.4100a Chronic To xicity (rodent) ...........................................

870.4100b Chronic Toxic ity (nonro dent) ...................................... 870.4200a Oncogeni city (rat) ....................................................... 870.4200b Oncogenic ity (mouse) .................................................

870.4300 Chronic/On cogenicity ................................................. yes

yes

yes

yes

yes yes 3

yes 3

yes

yes

yes

870.5100 Mutagenicity—Gene Mutation - bacterial .................. 870.5300 Mutagenicity—Gene Mutation - mammalian ............. 870.5375 Mutagenicity—Structural Chromosomal Aberrations. 870.5550 Mutagenicity—Other Genotoxic Effects ..................... yes

yes

yes

yes yes

yes

yes

yes

870.6100a Acute Delayed Neurotoxicity (hen) ............................ 870.6100b 90-Day Neur otoxicity (hen) ........................................ 870.6200a Acute Neurotoxicity Screening Battery (rat) ............... 870.6200b 90-Day Neur otoxicity Screening Battery (rat) ............ 870.6300 Develop. Neurotoxic ity ............................................... no

no

yes

yes

no --

-- yes

yes

--

870.7485 General Metabolis m .................................................... 870.7600 Dermal Penetra tion ...................................................... yes

no yes

no

870.7800 Immunotoxicity Series................................................... yes yesKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 35 of 60 TABLE A.1 Toxicology Data Requirement s for Kasugamycin, Technical.

Test Required Satisfied

Special Studies for Ocular Effects Acute Oral (rat) ...........................................................

Subchronic Oral (rat) .................................................. Six-month Oral (dog) .................................................. no

no

no --

--

--

1. Test material purity for the material used in these stud ies not provided in report, but was reported in a different study. 2. Study waived by HED, based on low acute toxicity, exposure potential, and high MOEs (HASPOC memo, TXR #0052461). 3. Satisfied by OCSPP Toxicity Test Guideline 870.4300.

A.2 Toxicity Profiles for Kasugamycin

TABLE A.2.1 Acute Toxicity Profile for Kasugamycin Technical Product.

Test Material 1
[% ai] Guideline

Number Study Type MRID

Number Results Toxicity Category

Technical product [81] 870.1100 Acute oral - rat 45910012 LD50 ( ♂ + ♀ ) > 5000 mg/kg IV 2

Technical product [81] 870.1200 Acute dermal-rat 47945757 LD50 ( ♂ + ♀ ) > 2000 mg/kg III 2

Technical product [84.8] 870.1300 Acute inhalation - rat 47945758 LC50 ( ♂ + ♀ ) > 4.892 mg/L IV

Technical product [84.8] 870.2400 Acute eye irritation -

rabbit 47945759 Mild eye irritant (iritis at 1 hour, resolving by 24 hours; conjunctivitis at 1 hour, resolving by 24 hours). III

Technical product [84.8] 870.2500 Acute dermal

irritation - rabbit 47945760 Not irritating to the skin. III

Technical product [84.8] 870.2600 Skin sensitization - guinea pig 47945761 Not a sensitizer under the conditions of this study. Not applicable.

1. Technical product Lot #KP-913 contains 81%, and Lot #KP-459 contains 84.8% kasugamycin hydrochloride hydrate. Not adjusted for the free acid. 2. Studies considered “supplementary/upgradable” due to lack of information on purity and characterization of test material. However, a stated purity of 81% for kasugamycin technical product Lot #KP-913 was located in

other studies.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 36 of 60 TABLE A.2.2 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 and kidney effects in females (increased urinary pH and epithelial cells in urine). Slight lipofuscin deposition in the proximal tubule cells were

observed at ≥ 1000 ppm in males but were not considered adverse).

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.

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.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 37 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

870.3200 21-Day dermal toxicity – rat

Acceptable/guideline 47945762 M: 0, 50, 250 or 500 mg/kg/day

F: 0, 50, 250/100 or 500/200 mg/kg/day, applied to the shaved skin; highest two dose

levels were decreased beginning on Day 8. Systemic

NOAEL ≥ 500 mg/kg/day (M), ≥ 500/200 mg/kg/day (F).

LOAEL - not determined. No effects observed at the highest dose

tested.

Dermal

NOAEL = 250 mg/kg/day (M), 250/100 mg/kg/day (F).

LOAEL = 500 mg/kg/day (M), 500/200 mg/kg/day (F), based on erythema, edema, eschar, ulceration, scabbing; microscopic findings of acanthosis, inflammation, and

ulceration.

870.3250 90-Day dermal toxicity Not required.

870.3465 90-Day inhalation toxicity Not required.

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; distention of the large

intestine with stool in the cecum.

Developmental

NOAEL = 1000 mg/kg/day

LOAEL = >1000 mg/kg/dayKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 38 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

870.3700 Pre-natal developmental in

nonrodents - rabbit

Acceptable/guideline 46030303 (main study)

46428701(rangefinding study) 0, 1, 3, 10 mg/kg/day (Main study)

Range finding study:

0, 10, 30, 100 mg/kg/day

Initial range finding study 0, 250, 500, 1000 – all doses of kasugamycin

resulted in severe maternal toxicity and

euthanasia. Maternal

NOAEL = 10 mg/kg/day

LOAEL = 30 mg/kg/day, based on the range-finding study results of maternal weight loss and decreased food consumption during treatment , abortion (1/4) on GD 22.

At 100 mg/kg/day – weight loss, abortions in 3 of 4 females, GD 18-

27. Developmental

NOAEL = 10 mg/kg/day

LOAEL = 30 mg/kg/day (rangefinding study) based on decreased mean fetal body weight

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 = 70.3/82.9 mg/kg/day (M/F)

LOAEL = 425.3/503.4 mg/kg/day (M/F), based on decreased body weights and body weight gains in P males, and red and swollen skin around the anal opening (M&F) (P and F1 animals).

Reproductive

NOAEL = 70.3/82.9 mg/kg/day (M/F)

LOAEL = 425.3/503.4 mg/kg/day (M/F) based on testicular atrophy/degeneration in F1 males, 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.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 39 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

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

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 μg/plate, with or without

S9 activation for Salmonella typhimurium strain G46 (his

- ).

0, 5, 10, 50, 100, 200 μg/plate, with or without

S9 activation 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.

870.5100 Gene mutation -

bacterial reverse mutation assay

Acceptable/guideline 47945764 0, 1.5, 5.0, 15, 50, 150, 500, 1500 or 5000 μg/plate, in the presence

or absence of S9

activation for Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and for

Escherichia coli strain

WP2 uvrA. No mutagenic activity in bacteria ( Salmonella typhimurium and

Escherichia coli ) under conditions of this assay.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 40 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

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 (CHO Cells)

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.

870.5375 Cytogenetics - in

vitro mammalian cell

chromosome aberration test (CHO cells)

Acceptable/guideline 47945765 0, 1085, 2170 or 4340 μg/ml, with or without S9

activation. No increase in mutant frequency, in the presence or absence of S9

activation.

870.5395 Cytogenetics

- mammalian erythrocyte micronucleus test,

mice

Acceptable/guideline 46030305 0, 200, 1000, 5000 mg/kg, with or without S9

activation. No evidence of induced chromosomal damage or other damage leading to micronucleus

formation.

870.5550 Other effects -

unscheduled DNA synthesis in cultured mammalian cells,

HeLa S3 cells

Acceptable/guideline 45910027 First assay: 0-2.5 mg/ml Second assay: 0-10 mg/ml Third assay: 0-10 mg/ml, both assays with or

without S9 activation. No evidence that unscheduled DNA synthesis was induced.

870.6200a Acute neurotoxicity screening battery -

rat (dose range-finding study)

Acceptable /nonguideline (rangefinding study) 47945766 0, 500, 1000 or 2000 mg/kg by gavage NOAEL = 2000 mg/kg

LOAEL not determined. No systemic or neurotoxic effects were observed at the highest dose tested.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 41 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

870.6200a Acute neurotoxicity screening battery -

rat

Acceptable/guideline 47945767 0, 500, 1000 or 2000 mg/kg by gavage NOAEL = 2000 mg/kg

LOAEL not determined. No systemic or neurotoxic effects were observed at the highest dose tested.

870.6200b 90-day neurotoxicity screening battery -

rat

Acceptable/guideline 47945768 0, 300, 3000 or 6000 ppm

in the diet

M: 0, 21, 210 or 439 mg/kg/day, males;

F: 0, 23, 238 or 486 mg/kg/day, females Systemic

NOAEL = 210/238 mg/kg/day (M/F)

LOAEL = 439/486 mg/kg/day (M/F), based on decreased body weight and body weight gain.

Neurotoxicity

NOAEL ≥ 439/486 mg/kg/day.

LOAEL not determined. No

neurotoxic effects were observed at the highest dose tested.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 42 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

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 ur ine, 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).Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 43 of 60 TABLE A.2.2 Subchronic, Chronic and Other Toxicity Profile for Kasugamycin.

Guideline

Number Study Type/ Classification MRID

Number Doses Results

870.7800 Immunotoxicity -

female mice

Acceptable/guideline 48132601 0, 300, 3000 or 10,000/7000 ppm in the diet for 28 days

AFC group: 0, 70, 755 or 2468 mg/kg/day; NK group: 0, 68, 691 or 2531 mg/kg/day (high

dose reduced to 7000 ppm on Day 16) Systemic

NOAEL = 755/691 mg/kg/day (AFC group/NK group)

LOAEL = 2468/2531 mg/kg/day (AFC group/NK group), based on clinical observations, decreased mean body weights and decreased hematological parameters.

Immunotoxicity

NOAEL ≥ 2468/2531 mg/kg/day (AFC group/NK group)

LOAEL not determined. A significant decrease (p ≤ 0.05) in Total Spleen Activity (AFC/spleen) was only observed at a dose causing excessive toxicity (HDT) and that

exceeded the limit dose.

A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Reference Dose (aRfD) - All Popul ations (Including Infants and Children, and Females Age 13-49 )

An acute reference dose was not selected becaus e an appropriate endpoint resulting from a single exposure was not identified. No maternal or deve lopmental effects were observed in the rabbit developmental toxicity study up to 10 mg/kg/day. In the dose range-finding study for the rabbit developmental toxicity study, maternal weight loss was observed at 30 mg/kg/day during dosing, along with decreased food consump tion, but is not considered an acute effect. An abortion in one female at 30 mg/kg/day occurred following the weight loss on GD 22, and was therefore not considered a single-dose effect. Abortions at 100 mg/kg/day occurred on GD 18 or later following weight loss. No eff ects were observed in the rat acu te neurotoxicity screening study, which tested up to a limit dose of 2000 mg/kg.

A.3.2 Chronic Reference Dose (cRfD)

Study selected: Combined chronic toxicity/carci nogenicity in the rat (oral).

MRID numbers: 45910024

Dose and endpoint for risk assessment: NOAEL = 11.3 mg/kg/day, based on testicular atrophy and softening in male rats at the LOAEL of 116 mg/kg/day.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 44 of 60 Chronic RfD = 11 mg/kg/day (NOAEL) = 0.11 mg/kg/day 100 (UF)

Comments about study/endpoint/uncertainty factors: The rat chronic toxicity/carcinogenicity study was selected for the cRfD because it was of the appropriate route and duration and is protective of effects seen in othe r studies. Although the testicular findings were not statistically significant compared to controls, these effects we re selected as the basis for the study LOAEL because the incidence and severity were increase d, and similar degenerative testicular effects were seen across species.

Selection of this endpoint is supported by the rat two-generati on reproductive toxicity study, in which a comparable parental NOAEL of 13.7 mg/ kg/day was observed. The parental LOAEL is 70.3 mg/kg/day, based on statisti cally significant decreased body weights/weight gains in P generation males, but no offspring toxicity wa s observed. Testicular at rophy, along with reduced male and female fertility and fecundity indices a nd increased precoital interval for the mating to produce the F2b litter, were observe d at 425.3/503.4 mg/kg/day (M/F).

The selected endpoint is also pr otective of maternal and develo pmental toxicity (weight loss and decreased food consumption during dosing, abortion in one female, decreased fetal weight) that was seen in the rabbit developm ental toxicity range-finding study. No developmental toxicity was seen in the rat and maternal toxicity was seen at 1000 mg/kg/ day (200 mg/kg/day).

A.3.3 Incidental Oral Exposure (Short- and Intermediate-Term)

An endpoint for incidental oral exposure was not selected because there are no proposed or existing residential uses for kasugamycin.

A.3.4 Dermal Exposure (Short- and Intermediate-Term)

A short- and intermediate-term dermal exposure assessment is not required because an appropriate endpoint was not identified. No syst emic toxicity was observed in the rat 21-day dermal toxicity study at doses th at caused local dermal irritati on by one week of exposure. The selected dose and endpoint are protective of ot her endpoints of concern (decreased body weight, testicular or renal toxicity, gastro intestinal or oral cavity effects) because they were evaluated in the dermal study and there are no concer ns for developmental susceptibility.

A.3.5 Dermal Exposure (Long-Term)

A long-term dermal occupational risk assessmen t is not required because long-term dermal exposure is not anticipated from the proposed uses.

A.3.6 Occupational Inhalation Exposure, Sho rt-Term (1 to 30 Days ) and Intermediate-Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 45 of 60 Term (1 to 6 Months)

Studies selected: Co-critical subchronic toxicity study in dogs (oral) and developmental toxicity study in rabbits (oral) and its range-finding study.

MRID numbers: 46060302, dog subchronic study (lead study); 46030303, main study, and 46428701, range-finding study, rabbit developmental study.

Dose and endpoint for risk assessment: NOAEL = 11 mg/kg/day from the dog subchronic toxicity study, based on swollen mouth, tongue lesions, excessive salivation, thickened skin at

the commissures of the mouth and few feces. Supported by the maternal toxicity NOAEL of 10 mg/kg/day (main study HDT) from the rabbit devel opmental toxicity stu dy, based on maternal weight loss and decreased food consumption during dosing, abortion ( GD 22) and decreased fetal weight at 30 mg/kg/day in th e rabbit range-finding study. Comments about study/endpoin t/uncertainty factors: Since an inhalation toxicity study was not available, oral toxicity studies were used to select a dose and endpoint. The selected doses and endpoints are the most sensitive identified for subchroni c exposure to kasugamycin. The studies are of the appropriate duration fo r short- and intermediate-term exposure, and are protective of potential developmental toxicity. These studies we re selected to be protective of potential effects to the respiratory tract as well as systemic toxicity, since kasugamycin shows evidence of irritation to the oral cavity and the gastrointestinal mucosa, as well as the skin (as seen in the rat 21-day dermal study). In the subchronic dog study, lesions of the oral cavity and tongue were considered portal of entry ir ritation effects. In the de velopmental rat study and the developmental range-finding study in the rabbit, gas distension of th e stomach, large intestine, or cecum were observed; rabbits were more sensitiv e than rats. Kasugamycin is poorly absorbed and metabolized, and is excreted primarily in th e feces as unchanged parent compound: the GI tract findings in these studies ma y therefore reflect the presence of irritation by the unabsorbed test material. A 28- or 90-day inhalation toxicity study is not required, ba sed on low use rate and anticipated inhalation exposure, low acute inhalation toxicity, lo w vapor pressure, and lack of persistence of kasugamycin in the environment: exposure via volatilizat ion is expected to be low. An additional uncertainty factor for lack of inhalation toxicity data (UF DB ) is therefore not needed. In the absence of inhalation data, inhalati on toxicity is assumed to be equivalent to oral study for the purpose of route-to-route extrapolation.

A.3.7 Inhalation Exposure (Long-Term)

A long-term inhalation occupationa l risk assessment is not require d because long-term inhalation exposure is not anticipated from the proposed or existing uses.

A.4 Executive Summaries (Studies Subm itted since Previous Assessments)

A.4.1 Subchronic ToxicityKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 46 of 60 870.3100 90-Day Dietary Toxicity – Rat (updated summary)

In a 90-day oral toxicity study (MRID #45910020), kasugamyci n hydrochloride hydrate (64.5% ai; Lot #KP-834) was administered to 12 Wistar ra ts/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 th e doses were adjusted for comp ound 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 lo wer (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 signifi cantly lower (5%) than controls (p  0.05) only on Week 6; however, non-significant decreases in body weight gain were observed throughout the study, resulting in a decrease of 8% in overall body weight gain. Although not considered treatment-related, significant (p  0.05) decreases in food consumpti on 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 were observed in females. Urine pH was decreased in both sexes (p  0.05) at 6000 ppm and also in females at 3000 ppm; this is possibly due to the acidic prop erties of the test co mpound. The urinalysis findings in females at 6000 ppm were considered evidence of possible urin ary tract toxicity.

Microscopically, at 6000 pp m in males, increased (p  0.01) incidences of eosinophilic bodies in the kidney proximal tubular cells (slight severity ) were observed (11/12 treated vs. 0/12 controls,

p  0.01). These lesions were also found in the 1000 and 3000 ppm males (7/11 and 11/12,

p  0.01). Foam cell aggregation in lungs of females (all of slight severity except 1 treated of moderate severity, 10/12 tr eated vs. 3/12 controls, p  0.01) were also reported. While related to treatment, the increased incide nce of both of these effects wa s not considered biologically significant due to the low severity and l ack of associated adverse findings.

No adverse effects were obser ved at doses equal to or lo wer than 3000 ppm. Although an apparent decrease in water consumption wa s seen in the 3000 ppm males, mean water consumption data was based on individual cage values, and no baseli ne water consumption measurements were taken prior to dosing. Theref ore, there is not enough information to make a

definitive evaluation of a treatment-related eff ect on male water consumption. Decreases in hematocrit, hemoglobin, and erythrocyte coun ts at 3000 and 6000 ppm were minor and not considered biologically significan t. The increased incidences of increased eosinophilic bodiesKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 47 of 60 (of slight severity) in the kidneys in males at ≥ 1000 ppm and foam cell aggregation (slight) in the lungs in the females at ≥ 1000 ppm were considered treatment -related but not toxicologically significant due to their low severity grade and lack of associated findings. This conclusion is supported by the rat combined chronic toxi city/carcinogenicity study (MRID #45910024). At 3000 ppm, the highest dose tested, an increased incidence of slight to moderate lipofuscin deposition was observed in kidney proximal tubul e cells, with no eviden ce of alterations to urinalysis/clinical chemistry parameters, kidney weight, or other kidney histopathology. Foam cell accumulation in lungs of males and females wa s also observed at 3000 ppm with no apparent adverse consequences.

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, and on urinalysis findings in females suggesting toxicity to the ki dney. 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.

870.3200 21-Day Dermal Toxicity - Rat

In a 21-day dermal toxicity study (MRID 47945762) in rats, kasuga mycin technical (71.5% a.i., free base), Batch No. KMB 0808) was applied to th e clipped skin of 10 animals/sex/dose. The test substance was applied at dose levels of 0 (vehicle control) , 50, 250, or 500 mg/kg/day (males) and 0, 50, 250/100, or 500/200 mg/kg/day (f emales) over a 6-hour exposure period daily for 21 consecutive days. In females, because of severe dermal effects observed on Day 7, the dose levels in the 250 and 500 mg/kg/day test groups were reduced to 100 and 200 mg/kg/day, respectively, on Day 8 and for the re mainder of the treatment period.

Reddening and scabbing of the dose site were observed in both se xes in the mid- and high-dose groups beginning on Day 5 and Day 7 for females a nd males, respectively. These observations generally persisted in a dose-dependent manne r throughout the treatment period. On Day 7, 40% and 90% of the females in the mid- and high-dose groups, respectively, had very slight to severe erythema and/or edema (dose-dependent) at the dose site; eschar and coriaceousness were observed in the high-dose group. Following redu ction of the dose in the mid- and high-dose female groups, the frequency and severity of the dermal findings decreased in the mid-dose group, while the effects in the high -dose group varied in severity (i ncreased, decreased, or were unchanged). On Day 7, 40% of the males in the high-dose group showed very slight to slight erythema and/or edema at the dose site. The fr equency and severity of these observations were reported to increase over the course of the treatm ent; with 80% of the male s showing very slight to severe erythema and/or edema by Day 21; esch ar and encrustation also were observed in the high-dose group males by Day 21. Mi croscopic observations of treat ed skin showed 90% of the males and 70% of the females in the high-dose gr oups had acanthosis, acute inflammation and/or

ulceration at the dose site at the end of the treatment peri od. No test-substance-relatedKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 48 of 60 microscopic findings were seen in either sex in the low- and mid- dose treatment groups. No macroscopic test substance-related dermal findi ngs were reported for the low-dose groups.

The LOAEL for dermal toxicity for kasugamycin technical (expressed as active ingredient) in rats is 500 mg/kg/day for males and 500/ 200 mg/kg/day for females, based on adverse dermal lesions at the dose site (eryth ema, edema, eschar, encrustation, ulceration, coriaceousness, and/or scabbing), with correl ating microscopic findings (acanthosis, acute inflammation, ulceration and/or granul omatous inflammation) in both sexes. The NOAEL for dermal toxicity for kasu gamycin technical (expressed as active ingredient) is 250 mg/kg/day for males and 250/100 mg/kg/day for females.

There were no effects on clini cal observations or on mean body weight, food consumption, absolute and relative organ weights, hematological parameters, clinical chemistry, or urinalysis

determinations in either sex at any dose level associated with e xposure to kasugamycin technical. A few microscopic observations in animals in th e high-dose groups (mild my eloid hyperplasia in

the sternal and femoral bone ma rrow of one male, minimal lym phoid necrosis of the thymic

cortex and minimal vacuolat ion of the adrenal cortex in one male, and minimal lymphoid necrosis in the thymus of 2 females) were consid ered to be secondary to dermal inflammation or stress responses to dose application and handlin g procedures, and not directly test substancerelated. Statistically significant differences in some hematological parameters in males were

attributed to individual outli er values resulting from tissue inflammation or necrosis not

associated with test substance treatment. A 5.4% decrease in the mean body weights of males in the high-dose group (not statistica lly significant) compared with co ntrols was not considered to

be treatment-related. There was over a 22% decr ease in body weight gain in males and over a 19% decrease in females at the highest dose test ed. However the severe irritation at this dose lessens the biological signifi cance of this finding. Three uns cheduled mortalities occurred during the treatment period (one control male, one low-dose male, one mid-dose female); these

deaths also were not considered to be treatment-related.

The NOAEL for systemic effects for kasugamycin technical (expressed as active ingredient) is greater than 500 mg/kg/day for males and greater than 500/200 mg/k g/day for females, the highest tested doses, based on body weig ht, food consumption, absolute and relative organ weights, clinical observations, hemato logical parameters, clinical chemistry, and urinalysis determinations. A LOAEL for syst emic effects could not be determined (i.e., greater than the highest tested doses).

This 21-day dermal toxicity study in the rat is acceptable/guideline and satisfies the guideline requirement for a 21-day dermal toxi city study (OPPTS 870.3200) in rats.

A.4.2 Developmental toxicity

870.3700b Developmental Toxicity – Rabbit (updated summary)Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 49 of 60 In a developmental toxicity study (MRI D #46030303), kasugamycin hydrochloride hydrate (62.7% ai, Lot #KP-570) in distille d 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 doe s were killed on GD 29; their fetuses were removed by cesarean section and examined. Dosi ng was based on the results of a range-finding study (MRID 46428701). Initially doses of 0, 250, 500 or 1000 mg/kg/day were administered to 4 pregnant rabbits/dose group, but due to excessive maternal toxicity, additional groups were added at 0, 10, 30 and 100 mg/kg/day.

Maternal toxicity : In the main study, there were no tr eatment-related effects on maternal mortality, clinical signs, body weight, food consumption or gr oss pathology. Although effects were not seen at the highest dose tested, a new de velopmental 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 study. At 10 mg/kg/ day, one female was sacrificed in extremis on GD 27 but gross examination at necropsy showed evidence of a gavage error. At 30 mg/kg/day, one female was found dead on GD 11, but this animal also show ed evidence of gavage error. Decreased maternal body weight (up to 10% below controls) due to weight loss, primarily in the first week of dosing (from GD 6-14, control to high dos e 0.11, 0.02, -0.08 and -0.38 kg, respectively) were observed. Food consumption was also lower dur ing the early portion of treatment. One doe aborted on GD 22; this was consider ed secondary to the body weight effects. At 100 mg/kg/day,

one female was sacrificed in extremis on GD 21

– this death was consid ered treatment-related, based on lack of evidence of gavage error at necropsy. Body weight loss was marked during dosing and 3 of 4 animals aborted on GD 18, 19 a nd 27, secondary to the weight effects. All

animals administered doses of ≥ 250 mg/kg/day in the initial dosi ng groups were sacrificed on GD 14 to 16 due to sharply reduced food consump tion (<10% of controls) and weight loss (body weight decreases exceeding 10% relative to GD 6 wei ghts). Females that died or were sacrificed during the study showed fluid and gaseous di stension of the gastro intestinal tract. The maternal toxicity LOAEL is 30 mg/kg/d ay, based on weight loss and decreased food consumption during dosing and an abortion following we ight loss in the range-finding study. The maternal toxicity NOAEL is 10 mg/kg/day based on lack of ef fects in the main study and the range-finding study.

Developmental toxicity : In the main study, there were no trea tment-related effects on fetuses. In the range-finding study, at 30 mg/kg/day feta l mean body weight was reduced (15% below controls) in the two litte rs that were available for evaluation. Due to mortality and abortions at

doses ≥ 100 mg/kg/day, no litters were available for examination. No treatment-related fetal abnormalities were reported. The developmental toxicity LOAEL is 30 mg/kg/day, based on decreased mean fetal weight in the range-fi nding study. The developmental NOAEL is 10 mg/kg/day, based on lack of effects in the main study and the range-finding study.

This study is classified acceptable/guideline and satisfies the guideline requirements for a developmental toxicity study (OPPTS 870.3700b; OECD 414) in the rabbit, when the main study is considered together with the range-finding study.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 50 of 60 A.4.3 Reproductive toxicity

870.3800 Reproductive Toxicity – Rat (updated summary)

In a two-generation reproduction toxicity study (MRID #45910023), kasuga mycin 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 leve ls of 0, 200, 1000, or 6000 pp m (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/s ex/group (1 pup/sex/litt er 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 we ights 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 cumula tive body weight gains we re decreased in the 1000 ppm males for Weeks 3 to 9 (8%; p  0.05) and in the 6000 ppm males for Weeks 3 to 14 (11%; 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 (28% below cont rols, week 0-11; p  0.01). Based on the marginal body weight and weight gain decreases relative to controls (<10%) obs erved at 1000 ppm in only one parental generation, the decreases observed at this dose, although trea tment-related, were not considered adverse.

Red and swollen skin around the anal opening was observed exclusively in the 6000 ppm group in both sexes and in both generations. This clin ical 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 bot h sexes and both generati ons (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 in flammation and ulceration of the rectum were observed in both sexes of both generations (88 to 100% treated vs 0 controls). Additionall y, in the P males at this dose, squamous cell hyperplasia was observed at the ano-rectal j unction (38% treated vs 0 controls).

At 6000 ppm, testes that were small (38%) and containing fluid (67%) we re observed (compared to 0% of controls) in the F 1 males at necropsy. Microscopica lly, 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 byKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 51 of 60 complete loss of the germinal epithelium, atr ophy of the seminiferous tubules, persistence of Sertoli cells, and variable amounts of intert ubular 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 6000 ppm (equivalent to 425.3/503.4 mg/kg/day [M/F]), based on decreased body weights and body weight gains in the P and F1 generation males, increased incidence of pelvic dilatation of the kidney and chronic progressive nephropathy in F1 males, and lesions of the anal opening in P and F1 animals of both sexes. The NOAEL is 1000 ppm (equivalent to 70.3/82.9 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 lactatio n period. Furthermore,

there were no treatment-relate d 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, or gan weights, and histopathology.

Based on the parameters investigated, the LOA EL 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-coita l 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 wa s 6000 ppm (equivalent to 425.3/503.4 mg/kg/day [M/F]) based on testicular atrophy in P and F1 males, decreased fertility and fecundity in

the F 1 parents for both litters and increased pre- coital interval during the mating period

for the F 2 litter. The NOAEL is 1000 ppm (e quivalent 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-generati on reproduction study in the rat.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 52 of 60 A.4.4 Neurotoxicity

870.6200a Acute Neurotoxicity Screening Study - Rat

In an acute neurotoxicity study (MRID # 47945767) , Kasugamycin techni cal (purity 71.5% free base, Lot no. KMB-0808) was administered at doses of 0, 500, 1000 or 2000 mg/kg body weight

dissolved in deionized water to Crl:CD(SD) rats (12 rats/sex /dose) by oral gavage. The dose volume was 10 mL/kg for all groups. All animals were observed twice daily for mortality and morbidity, clinical observation recorded daily and body weights recorded weekly. A functional observational battery (FOB) and locomotor activ ity evaluations were performed prior to exposure (pretest on Day -7), approximately 1 hour following dose administration (time to peak effect) on study Day 0, and on Days 7 and 14. At st udy termination, 6 animals/sex in the control and 2000 mg/kg groups were euthanized and perfused in situ for neuropathological evaluations. Brain weights and measurements were recorded for all animals and central and peripheral nervous system tissues were examined for microscopic changes.

No deaths occurred in any of the four groups of animals, nor were there any Kasugamycinrelated effects on clinical condi tion, body weights or body weight ga ins. No test substance dose related effects were recorded in brain weight s or brain dimensions in perfused animals. Functional observational battery (FOB) and total motor activity revealed no treatment-related effects in male and female rats at any dos e levels of 500, 1000, and 2000 mg/kg on study Days 0, 7, and 14. No dose related macroscopic finding we re observed in male and female rats at necropsy. At 2000 mg/kg, no treatmen t-related histological changes were observed in the central or peripheral nervous system tissues.

The results of the current study suggest that the NOAEL for neurotox icity of Kasugamycin is 2000 mg/kg body weight in the rat. The LOAEL is not determined.

This study is classified as acceptable and satisfies the guideline requirement (870.6200; OECD 424) for neurotoxicity screen ing battery in rats.

870.6200b Subchronic Neurotoxicity Screening Study - Rat

In a 90-day dietary neurotoxicity study (M RID # 47945768), Kasugamycin technical (purity 71.5% free base, Lot no. KMB-0808) was administered continuously in the diet at doses of 0, 300, 3000 or 6000 ppm to Crl:CD(SD) rats (12 rats/s ex/dose) for at least 90 consecutive days (approximately 13 weeks). All animals were obser ved twice daily for mortality and morbidity. Clinical observations, body weights, and food c onsumption were recorded weekly. A functional observational battery (FOB) and locomotor activ ity evaluations were performed prior to exposure (pretest on study week -1) and duri ng study weeks 1, 3, 7, and 12. At study termination, 6 animals/sex in the control and 6000 pp m groups were euthanized and perfused in situ for neuropathological evaluations. Brai n weights and dimensions were recorded for all animals andKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 53 of 60 central and peripheral nervous system tissues were examined for microscopic changes.

No deaths occurred in any of the four groups of animals, nor were there any Kasugamycinrelated effects on clinical condi tion during the weekly detailed physical examinations. No testsubstance-related effects were apparent on mean male and female rats food consumption at any exposure levels. There was a significant (p <0.05 or p<0.01) reduction in mean male body weights in the 6000 ppm group during study week s 2-13 whereas mean female body weights were significantly (p<0.05) reduced on study w eek 4 only compared to control group. In addition, mean body gains were significantly (p <0.01) reduced during study weeks 1-2, 3-4, and 4-5 for the males and study week 3-4 for the females following Kasugamycin exposure. Mean body weights for the male rats in the 300 and 3000 ppm groups were consistently lower and showed an exposure related trend, however they we re not statistically different from controls. The average calculated test substance consum ption (intake) was 21, 210, and 439 mg/kg/day for males and was 23, 238, and 486 mg/kg/day for fema les. Functional observational battery (FOB) and mean ambulatory and total motor activity reveal ed no effects in male and female rats at any dose levels of 300, 3000, and 6000 ppm on study w eeks 1, 3, 7, and 12. No test substance dose related effects were recorded in brain weights or brain dimensions in perfused animals. No dose related macroscopic finding were observed in ma le and female rats at necropsy. At 6000 ppm, no treatment-related histological changes were obser ved in the central or pe ripheral nervous system

tissues.

The results of the current study suggest th at the NOAEL for subc hronic toxicity of Kasugamycin is 3000 ppm for males (210 mg /kg body weight/day) and females (238 mg/kg/day). The LOAEL is 6000 ppm, based on decreased body weight and body weight gain. The NOAEL for subchronic neurotoxicity is 6000 ppm for males (439 mg/kg/day) and females (486 mg/kg/day).

This study is classified as acceptable and satisfies the guideline requirement (870.6200; OECD 424) for neurotoxicity screen ing battery in rats.

A.4.5 Mutagenicity

870.5100 Bacterial Reverse Gene Mutation

(1) In a toxicity study (MRID 47945764), kasugamycin (active ingredient/f ree base purity of 71.5%) was tested in th e Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvr A in the presence and absence of Aroclor-induced rat liver S9.

The assay had two phases, using the plate incorp oration method. The first phase, the initial toxicity-mutation assay (Experiment B1), wa s used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 54 of 60 The second phase, the confirmatory mutagenicity assay (Experiment B2), was used to evaluate and confirm the mutagenic potential of the test article. Dosing formulations were adjusted for purity using a correction factor of 1.4 (50 mg kasugamycin x 1 mg kasugamycin technical/0.715 mg kasugamycin active ingredient = 70 mg kasugamycin technical).

The test article formed a soluble and clear solution in water at approximately 50 mg/mL kasugamycin technical, the maximum concentra tion tested in the solubility test.

The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μ g kasugamycin active ingredient per plate. Maximum dosage was ach ieved using a concentration of 50 mg/mL and

100 μ L plating aliquots. In the initial toxicity-mut ation assay, no positive mutagenic response

was observed.

In the confirmatory mutagenicity assay, no pos itive mutagenic response or precipitate was observed. Toxicity was obser ved beginning at 500 or 1500 μ g per plate.

Positive controls induced the appropriate responses in the presence and absence of S9 activation.

The results of this study indicate the test ar ticle, Kasugamycin technical, was concluded to be negative in the Bacterial Reverse Mutation Assay.

This study was conducted in compliance with the te sting guidelines of the OECD (1998) and the

US EPA Health Effects Guid elines OPPTS 870.5100 (1998).

870.5375 In Vivo Mammalian Cytogenetics

Kasugamycin, Technical, was tested in th e chromosome aberration assay (MRID 47945765) using Chinese hamster ovary (CHO) cells in bot h the absence and presence of an Aroclorinduced S9 activation system. A toxicity test was performed to establish a dose range for the chromosome aberration assay. The chromosome aberration assa y was used to evaluate the clastogenic potential of the test substance. The dosing preparations were adjusted for the purity (71.50%) of the test substa nce using a correction f actor of 1.4. Cytotoxicity in the preliminary and definitive chromosome aberration assays was determined by relative increase in cell counts.

Water was the solvent and the test substance wa s soluble in water at a concentration of 43.4 mg/mL, the maximum concentration prepared in the assay.

In the preliminary toxicity assay, the maximum dose tested was 4340 μ g/mL (10 mM). The test

substance was soluble in water and in the treatme nt medium at all dose levels tested at the beginning and conclusion of the treatment period. Substantial toxicity (i .e., at least 50% cell growth inhibition, relative to the solvent control) was not observed at any dose leve l in all three

exposure groups.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 55 of 60 In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9-act ivated test system. All cells were harvested 20

hours after treatment initiation. The test substa nce was soluble in wate r and in the treatment

medium at all dose levels tested at the beginning and conclusion of the treatment period. In the absence of both test substance precipitation in the treatment medium and at least 50% toxicity, the highest dose level evaluated was 4340 μ g/mL Two additional lower dose levels were included in the evaluation. The perc entage of cells with structural or numerical abe rrations in the test substance-treated groups wa s not significantly increased relati ve to solvent control at any dose level (p>0.05, Fisher's Exact test).

The results of this study indicate the test ar ticle, Kasugamycin Technical, was negative for

the induction of structural and numerical ch romosome aberrations in CHO cells in both

non-activated and the S9-act ivated test systems.

This study was conducted in compliance with the testing guidelines of the OECD 473 (1998) and

the US EPA Health Effects Gu idelines OPPTS 870.5375 (1998).

A.4.6 Immunotoxicity

870.7800 Immunotoxicity - Mouse

In a 28-day dietary immunot oxicity study (MRID 48132601), kas ugamycin technical (71.5% free base/a.i., Batch No. KMB-0808) was administ ered to two study groups (AFC and NK) of

female Crl:CD-1 mice in the diet at concentrat ions of 0 (vehicle and positive control groups), 300, 3000 or 10,000 ppm (10 animals/ dose/study group) as the acti ve ingredient, for 28 consecutive days. Because of severe weight loss in the AFC and NK groups dosed with 10,000 ppm, the dietary concentration in the hi gh-dose groups was reduced from 10,000 ppm to 7000 ppm on Day 16 (AFC group) or Da y 15 (NK group). The average daily dose of kasugamycin (a.i.) was 0, 70, 755, or 2468 mg/kg/day fo r the AFC dose groups, and 0, 68, 691, or 2531 mg/kg/day for the NK dose groups.

On Day 24, animals in the AFC study group received a single dose of sheep red blood cells (1 x

10 8 SRBCs in 0.2 mL of Earl’s Balanced Salt Solution with HE PES buffer solution) via a tail vein, with the exception of one animal in the 10,000/7000 ppm group that received an intraperitoneal injection. Anim als in the AFC positive control group also were administered cyclophosphamide monohydrate by intr aperitoneal injection once da ily on Days 24-27 at a dose rate of 50 mg/kg/day. Animals in the AFC study group were used to evaluate the effect of kasugamycin technical on the T-cell dependent antibody response (humoral immunity). Animals in the NK positive control group were dosed with a single intravenous inje ction (0.2 mL) of antiasialo GM1 formulation (1:10 dilution of a reconstituted anti-asialo GM1 vial) on Day 27. Animals in the NK study group were used to eval uate the effect of kasugamycin technical on innate immunity.Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 56 of 60 Three animals in the high-dose AFC group were euthanized in extremis on Days 21, 23, and 25, respectively, because of significant body weight losses and clinical findings. There were no

other unscheduled mortalities in any other AFC dose group or at any dose level in the NK study group. Animals in the both the AFC and NK hi gh-dose groups had significantly decreased (p ≤ 0.01) mean body weights during Weeks 2, 3, and 4; decreased mean cumulative body weight gains during Weeks 0-2, 0-3, and 0-4; and de creased mean terminal body weights when compared with the vehicle control. There we re no test substance treatment-related effects on food consumption in the low- and mid-dose AF C and NK groups. The effect of the test substance on food consumption by the high-dose AF C and NK groups could not be determined.

Test substance treatment- related changes in he matological parameters were seen in the highdose NK group. Mean corpuscular hemoglobin and mean corpuscular volume were significantly lower than the control values (p ≤ 0.01 and p ≤ 0.05, respectively); mean hemoglobin distribution width and mean absolute and percent ne utrophil counts were significantly higher (p ≤ 0.01) than control values. Hematological changes seen in the mid-dose group, including decreases in mean corpuscular hemoglobin and mean corpuscular volume, and increases in mean hemoglobin distribution width (not statistica lly significant), and statisticall y significant increases in mean

total white blood cell and ab solute lymphocyte counts (p ≤ 0.05 and p ≤ 0.01, respectively) were considered to be possibly related to test substa nce treatment. There were no test substancetreatment-related effects on hematological para meters in the low-dose NK group. Hematological parameters were not evalua ted in the AFC study group.

There were no test substance treatment-related gross macroscopic findings reported for any AFC or NK treatment group. There were no statistically significant effect s of test substance treatment on absolute and relative mean spleen and thym us weights in any of the NK dose groups. A significant decrease in mean absolute spleen weight (p ≤ 0.05) was seen in the high-dose AFC group (absolute and relative thymus weights and relative spleen weights were not evaluated); however, the measurement of spl een weights of immunized animals was not considered to be the most appropriate way to evaluate the effect of a test substance on spleen weight.

For systemic toxicity related to treatment with kasugamycin technical, the LOAEL for female mice is 10,000/7000 ppm (highest tested dose, equivalent to 2468 and 2531 mg/kg/day active ingredient, fo r the AFC and NK groups, respectively), based on clinical observations, mean body weights, and mean hematological parameters (NK study group). The NOAEL is 3000 ppm (equivalent to 755 and 691 mg/kg/day active ingredient, for the AFC and NK groups, respectively) based on these parameters.

In the AFC assay (AFC study group), there were no statistically significant decreases in immune response in the test substance treatment groups when evaluated as Specific Activity (AFC/10 6
spleen cells). A statisti cally significant decrease (p ≤ 0.05) in Total Spleen Activity (AFC/spleen) was seen in the high-dose AFC group when compar ed with the control. Because the decreased mean body weights in the high-dose group exceeded the maximum tolerated dose, the results

were considered inconclusive regarding potential immunotoxicity. There were no statisticallyKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 57 of 60 significant test substance treatment-related e ffects on NK cell activity at any dose level (NK study group).

For immunotoxicity related to treatment with kasugamycin technical, t he NOAEL for female mice is 10,000/7000 ppm (highest do se tested, equivalent to 2468 and 2531 mg/kg/day active ingredient, for the AFC and NK groups, respectively) based on the overall weight-of-evidence: no effect on na tural killer cell activity (NK assay) and no statistically significant effect on absolute and relative thymus weights in the NK group; no effect on mean Specific Activity (AFC assay) , and inconclusive evidence of test substance treatment effects on mean Total Spleen Activi ty (AFC assay) and mean absolute spleen weights in the AFC group. The LOAEL fo r immunotoxicity is greater than 10,000/7000 ppm, highest tested dose.

This 28-day dietary immunotoxi city study in the mouse is acceptable/guideline and satisfies the guideline requirement for a 28-day immunot oxicity study (OPPTS 870.7800) in mice.

APPENDIX B: Metabolis m Summary Tables

TABLE B.1 Chemical Names and Stru ctures of Compound s Detected in the Goat Metabolism and Confined Rotational Crop Studies.

Common Name

Matrix Chemical Name Chemical Structure

Kasugamycin

Goat kidney, liver, and renal fat. Rotated lettuce, wheat forage, hay, straw, and grain. 3- O -[2-amino-4-[(carboxyiminomethyl)amino]-2,3,4,6-tetradeoxyα -D- arabino -hexopyranosyl]-Dchiro -inositol

Kasugamycinic

Acid

Wheat hay. N-[5-amino-2-methyl-6-(2,3,4,5,6-pentahydroxycyclohexyloxy)tetrahydro-pyran-3- yl]-oxallamic acid *

* Chemical name generated using naming software from ISIS/Draw (refer to D308573; W.T. Drew; 17 August 2005; PP#3E6579).Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 58 of 60 TABLE B.2 Chemical Names and Stru ctures of Compound s Detected in the Rat and Tomato

Metabolism Studies.

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) 3- O -[2-amino-4-[(carboxyiminomethyl)amino]-2,3,4,6- tetradeoxy- α -D- arabino

hexopyranosyl]-D- chiro

inositol

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

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 O

O OH OH

OH OH O H C H
3

N H NH

O H

O NH
2

O

O OH OH

OH OH O H C H
3

N H O

O H

O NH
2

O

O OH OH

OH OH O H C H
3

N H
2 NH
2



## . 2 HClKasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 59 of 60 TABLE B.2 Chemical Names and Stru ctures of Compound s Detected in the Rat and Tomato

Metabolism Studies.

Common Name [Code]

% Total Radioactive Residues Chemical Name Chemical Structure

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 [ 14 C]-kasugamycin to tomato plants at 0.17 lb ai/A (roughly 3X the proposed maximum seas onal 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 [ 14 C]-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.

APPENDIX C: Physical/Chemical Properties

TABLE C Physicochemical Properties of the Tech nical Grade Test Compound (Kasugamycin Hydrochloride Hydrate).

Parameter Value Reference

Molecular Weight 433.8 PP#3E6579 administrative materials (MRIDs #45910004 and -05) Melting Point/Range 202-230oC (decomposing)

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

Density 0.43 g/mL at 24.5 o 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 O

O OH OH

OH OH O H C H
3

N
H NH

O H

O NHCOCH
3Kasugamycin Human Health Risk Assessment DP Barcode No. D376608

Page 60 of 60 TABLE C Physicochemical Properties of the Tech nical Grade Test Compound (Kasugamycin Hydrochloride Hydrate).

Parameter Value Reference

Vapor Pressure <0.013 mPa at 25oC
Dissociation Constant (pK

a ) pK a1 = 3.23 pK a2 = 7.73 pK a3 = 11.0

Octanol/Water Partition Coefficient (Log [K OW ]) <1.96 at 23oC and pH 5

UV/Visible Absorption Spectrum Not available

APPENDIX D: Review of Human Research

This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical. Thes e data, which include studies from the Pesticide Handlers Exposure Database Vers ion 1.1 (PHED 1.1), the Agricultural Handler Exposure Task Force (AHETF) database, and the Agricultural Re-entry Task Force (ARTF) database are subject to ethics review pursuant to 40CFR §26, have received that review, and are compliant with applicable ethics requirements . For certain studies, that review may have included review by the Human Stud ies Review Board. Descriptions of data sources, as well as guidance on their use, can be found at: http://www.epa.gov/pesticides/science/handler-exposuredata.html and http://www.epa.gov/pesticides/sci ence/post-app-exposure-data.html

.