Document ID: EPA-HQ-OPP-2011-0641-0004
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2012-01-27T05:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                            WASHINGTON, D.C.  20460
                                                                      OFFICE OF
                                                            CHEMICAL SAFETY AND
\* MERGEFORMAT
                                                           POLLUTION PREVENTION

MEMORANDUM

Date:		December 2, 2011

SUBJECT:	Amicarbazone. Human Health Risk Assessment to Support Proposed Uses on Turf, Christmas Tree Farms, Non-Crop Areas and on Conifers in Nurseries.
 
PC Code:  114004 
DP Barcode:  D386858 
Decision No.:  441345
Registration No.:  66330-46
Petition No.:  NA
Regulatory Action:  Section 3 Registration
Risk Assessment Type:  Single Chemical Aggregate
Case No.:  NA
TXR No.:  NA
CAS No.:  129909-90-6
MRID No.:  NA
40 CFR:  180.615

FROM:	Linnea J. Hansen, Ph.D., Biologist
		Zaida Figueroa, Industrial Hygienist
		Douglas Dotson, Ph.D., Chemist
		Christina Swartz, Chemist
		Risk Assessment Branch II (RABII)
		Health Effects Division (7509P)

THROUGH:	Richard A. Loranger, Ph.D., Branch Senior Scientist
		Risk Assessment Branch II (RABII)
		Health Effects Division (7509P)

TO:		Michael Walsh/Kathryn Montague, RM Team 23
		Herbicide Branch
		Registration Division (7505P)

The Registration Division (RD) requested that the Health Effects Division (HED) conduct a risk assessment for the active ingredient amicarbazone to estimate the risk to human health that will result from proposed new uses on turf, ornamentals, Christmas tree farms, non-crop areas and on conifers in nurseries.  The attached human health risk assessment addresses exposure and risk associated with the proposed uses as well as the existing use on corn.  The exposures assessed include dietary (food and water), inhalation for occupational workers and residential handlers, toddlers' oral exposure from playing on treated turf, and aggregate exposure and risk for residential handlers and toddlers who play on treated turf.  There were no risks of concern identified for any route or duration of exposure.

1.0	Executive Summary	4
2.0	HED Recommendations	6
2.1	Data Deficiencies/Conditions of Registration	6
2.2	Tolerance Considerations	7
2.3	Label Recommendations	8
2.3.1	Recommendations from Occupational Assessment	8
2.3.2	Recommendations from Residential Assessment	8
3.0	Introduction	8
3.1	Chemical Identity	8
3.2	Physical/Chemical Characteristics	8
3.3	Pesticide Use Pattern	9
3.4	Anticipated Exposure Pathways	10
3.5	Consideration of Environmental Justice	10
4.0	Hazard Characterization and Dose-Response Assessment	11
4.1	Toxicology Studies Available for Analysis	11
4.2	Absorption, Distribution, Metabolism, & Elimination (ADME)	11
4.2.1	Dermal Absorption	12
4.3	Toxicological Effects	12
4.4	Safety Factor for Infants and Children (FQPA Safety Factor)	14
4.4.1	Completeness of the Toxicology Database	14
4.4.2	Evidence of Neurotoxicity	14
4.4.3	Evidence of Sensitivity/Susceptibility in the Developing or Young Animal	15
4.4.4	Residual Uncertainty in the Exposure Database	15
4.5	Toxicity Endpoint and Point of Departure Selections	16
4.5.1	Dose-Response Assessment	16
4.5.2	Recommendation for Combining Routes of Exposures for Risk Assessment	17
4.5.3	Cancer Classification and Risk Assessment Recommendation	17
4.5.4	Summary of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment	17
5.0	Dietary Exposure and Risk Assessment	19
5.1	Metabolite/Degradate Residue Profile	19
5.1.1	Summary of Plant and Animal Metabolism Studies	19
5.1.2	Summary of Environmental Degradation	20
5.1.3	Comparison of Metabolic Pathways	21
5.1.4	Residues of Concern Summary and Rationale	22
5.2	Food Residue Profile	24
5.3	Water Residue Profile	24
5.4	Dietary Risk Assessment	25
5.4.1	Description of Residue Data Used in Dietary Assessment	25
5.4.2	Percent Crop Treated Used in Dietary Assessment	26
5.4.3	Acute Dietary Risk Assessment	26
5.4.4	Chronic Dietary Risk Assessment	26
5.4.5	Summary Table	26
6.0	Residential (Non-Occupational) Exposure/Risk Characterization	27
6.1	Residential Handler Exposure	27
6.2	Postapplication Exposure	28
6.3	Combined Exposure	30
6.4	Residential Bystander Postapplication Inhalation Exposure	31
6.5	Spray Drift	31
7.0	Aggregate Exposure/Risk Characterization	32
7.1	Acute Aggregate Risk	32
7.2	Short-Term Aggregate Risk	32
7.3	Chronic Aggregate Risk	33
8.0	Cumulative Exposure/Risk Characterization	33
9.0	Occupational Exposure/Risk Characterization	33
9.1	Short-/Intermediate-Term Handler Risk	33
9.2	Short-Term Postapplication Risk	35
9.2.1	Dermal Postapplication Risk	35
9.2.2	Inhalation Postapplication Risk	36
10.0	References	36
Appendix A.  Toxicology Profile and Executive Summaries	37
A.1	Toxicology Data Requirements	37
A.2	Toxicity Profiles	38
A.3	Hazard Identification and Endpoint Selection	44
A.4	Executive Summaries	46
Appendix B.  Physical/Chemical Properties and Structures	68
Appendix C.  Review of Human Research	70

1.0	Executive Summary

HED has assessed the human health risk associated with proposed non-food uses of the herbicide amicarbazone.  Arysta LifeSciences North America, LLC requested use of amicarbazone for control of broadleaf weeds in turf (i.e., lawns, sod farms, golf courses, recreational fields, etc.), ornamentals, non-crop areas, Christmas tree farms and conifers in nurseries.  Amicarbazone is formulated as a 70% dry flowable powder, and is intended for pre-emergence and post-emergence applications.  Broadcast applications are to be made with ground equipment only, while spot treatments can be made with hand held equipment.  The product is most efficacious when application is followed by watering in or rainfall.

The toxicology database is complete, and is considered adequate for selecting toxicity endpoints for risk assessment.  The conditionally required subchronic inhalation study has not been submitted; HED is in the process of establishing criteria for determining when the study will be required, and may require a study for amicarbazone in the future.  The scientific quality of the database is relatively high, and the toxicity is well-characterized for a wide array of effects, including potential developmental, reproductive, immunologic and neurologic toxicity.  The major effects in mammals are general toxicity, as evidenced by decreased body weight and weight gain, and liver toxicity.  Decreased body weight and body weight gain were observed in studies with rats, mice and rabbits and were the most sensitive toxicological endpoints observed in many of the studies.  Slight effects on the thyroid gland were also seen in the subchronic rat and dog studies.   However, these effects were not observed in the corresponding chronic studies.  Additional mechanistic studies in rats did not show treatment related effects on thyroid weight or microscopic changes in the thyroid at any dose.

There was no evidence of increased quantitative or qualitative susceptibility in offspring in the available developmental and reproductive toxicity studies.  The delayed ossification observed in rat fetuses in the developmental rat study was not considered to be evidence of susceptibility since maternal toxicity was observed at similar doses, and because there was no corresponding decrease in fetal viability.  In the developmental rabbit study, maternal animals were more sensitive to amicarbazone than the developing fetuses.  In the 2-generation reproduction study, both maternal and developmental endpoints were based on decreased body weight and body weight gain.

Studies with amicarbazone also demonstrated the potential for neurotoxic effects, including a variety of clinical signs.  These effects were observed more frequently after gavage dosing in rats in the guideline studies as well as in several single gavage dose nonguideline studies; in addition, there was evidence that these effects were largely reversible.  No evidence of neurotoxicity was observed in the subchronic and developmental neurotoxicity studies in rats, which both involved dietary dosing.  The observed neurotoxicity in the acute neurotoxicity study was selected for acute dietary risk assessment; other endpoints selected for longer durations of exposure occurred at lower doses than those at which neurotoxicity was observed, and so the risk assessment is protective of potential neurotoxicity as well as the general toxicity observed throughout the database.  Similarly, the endpoints were selected to be protective of the potential immunotoxicity observed at the highest dose in the immunotoxicity study conducted in female rats.  There were no systemic effects observed in the 21-day dermal toxicity study in rats exposed to amicarbazone, and no treatment related signs of dermal irritation were observed.

The 10x FQPA factor has been reduced to 1x, and no additional uncertainty factors were retained for missing data.  This conclusion was based on the completeness of the database and the lack of increased quantitative or qualitative susceptibility.  Although neurotoxicity was observed in adult animals in gavage studies, no neurotoxic effects were observed in offspring in the rat developmental neurotoxicity study at dose levels that resulted in general systemic toxicity.   Potential immunotoxic effects were observed only at doses greater than those selected for risk assessment purposes and are therefore not of concern.  In addition, the exposure assessment is conservative and does not underestimate potential exposure and risk to young children or residential handlers.  The combined uncertainty factors of 100X (10X for intraspecies variability and 10X for interspecies extrapolation) serve as the level of concern (LOC) for occupational and residential exposure and risk.  Margins of exposure (MOEs) greater than 100 are not of concern.

Both the rat and mouse carcinogenicity studies indicate that amicarbazone is not likely to be carcinogenic to humans.  The mutagenicity battery for the chemical is complete and indicates that amicarbazone is not a mutagen.

Based on the submitted toxicology data, HED selected endpoints for acute and chronic dietary risk assessment, as well as for inhalation risk assessment for residential and occupational handlers.  There was no toxicity via the dermal route, and no concern for potential developmental effects; therefore, a dermal endpoint was not selected, and no dermal assessments were conducted.  Finally, HED selected an endpoint for incidental oral risk assessment, in order to assess toddlers' exposure to amicarbazone while playing on treated turf.  The acute dietary endpoint was clinical signs of neurotoxicity, while for all other scenarios endpoints were based on general toxicity (decreased body weight and body weight gains) and on liver and thyroid effects.  Inhalation risks were assessed using an endpoint from an oral study, and the assumption that inhalation toxicity would be equivalent to oral toxicity for the purpose of route-to-route extrapolation.

The dietary assessments included residues from the existing use on corn, and potential drinking water residues based on the turf application rate.  The highest potential drinking water exposure is from ground water sources.  Acute and chronic dietary exposure and risk are below HED's level of concern; the highest exposed population for both acute and chronic dietary was all infants <1 year old, at roughly 30% of the acute Population Adjusted Dose (aPAD) and 50% of the chronic PAD (cPAD).  The most significant exposure was from potential drinking water residues, which were very conservatively assessed.  Significant refinements to the food and drinking water assessments are possible but are not needed at this time to make the safety finding.

The occupational and residential assessments relied on standard assumptions with respect to body weight and area treated; in the absence of chemical specific studies, HED relied on unit exposures from surrogate data in accordance with HED's standard procedures.  Inhalation MOEs for residential handlers were significantly greater than the LOC of 100 (ranging from 3,600 to 220,000) and are not of concern.  For children playing on treated turf, incidental oral exposure resulted in an MOE of 910 for hand-to mouth exposure, 3,700 for object-to-mouth ingestion, and 270,000 for soil ingestion.  Residential exposures, when combined with exposure from food and drinking water, resulted in short-term aggregate risks that were not of concern, ranging from an MOE of 350 for toddlers' aggregate risk to an MOE of 1,100 for adult residential handlers.

Inhalation exposure and risk for occupational handlers treating the proposed use sites using a variety of equipment are not of concern, with MOEs ranging from 1,300 for mixing/loading for broadcast application to 1,500,000 for mixer/loader/applicator exposure associated with use of a backpack sprayer.

Postapplication inhalation exposure and risk were not quantitatively assessed, due to the low vapor pressure of the active ingredient and the use pattern, including the application rate and generally ground-directed and broadcast methods of application.  Further, residential and occupational handler inhalation assessments, considered protective of potential postapplication exposure and risk, did not result in risks of concern.

The risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical.  These studies were considered appropriate (or ethically conducted) for use in risk assessments.

2.0	HED Recommendations

HED has no objection to the proposed registration of the new uses of amicarbazone on turf, non-crop areas, Christmas trees, and on conifers in nurseries.

2.1	Data Deficiencies/Conditions of Registration

There are no data deficiencies that would preclude granting a full registration of the proposed non-food uses.

   * A subchronic (28-day) rat inhalation toxicity study is a conditional data requirement.  HED is in the process of establishing criteria for determining when the study will be required, and may require a study for amicarbazone in the future.  Nonetheless, HED's inhalation exposure and risk assessment is considered to be conservative and health protective. 

   * Additional storage stability data were requested to support the proposed use on field corn, and while the required data were submitted for field corn grain, forage and stover, additional storage stability data are needed for turnip roots and mustard greens, crops that could potentially be rotated with field corn.  These data are not likely to impact the risk assessment, since HED concluded the available data were adequate to establish tolerances for inadvertent residues in rotational crops, and since conservative assumptions were used in the dietary assessment.

2.2	Tolerance Considerations

The risk assessment addresses only the proposed non-food uses of amicarbazone on turf and ornamentals including Christmas trees.  There are no newly proposed tolerances, and no changes are recommended for the existing tolerance levels.  However, if additional food uses are to be proposed, the tolerance expression should be revised to reflect specific coverage and compliance statements in order for the tolerances to be properly enforced in accordance with the FFDCA.  The current tolerance expression is as follows:

      (a)       General.  Tolerances are established for the combined residues of the herbicide amicarbazone [4-amino-N-(1,1-dimethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and its metabolites DA amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and iPr-2-OH DA amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-hydroxy-1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide], calculated as parent equivalents, in or on the following raw agricultural commodities:
   
      (b)       Indirect or inadvertent residues.  Tolerances are established for the indirect or inadvertent residues of amicarbazone [4-amino-N-(1,1-dimethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and its metabolites DA amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and iPr-2-OH DA amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-hydroxy-1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide], calculated as parent equivalents, in or on the following commodities when present therein as a results of application of amicarbazone to the growing crops in paragraph (a) of this section:

The recommended revised tolerance expression should be incorporated into any additional new food use petitions:

   (a)	General.  Tolerances are established for the residues of the herbicide amicarbazone, including its metabolites and degradates, in or on the commodities in the table below.  Compliance with the tolerance levels specified below is to be determined by measuring only the sum of amicarbazone [4-amino-N-(1,1-dimethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and its metabolites desamino amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and isopropyl-2-hydroxy desamino amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-hydroxy-1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide], calculated as stoichiometric equivalents of amicarbazone, in or on the commodity.
   
   (b)	Indirect or inadvertent residues.  Tolerances are established for the indirect or inadvertent residues of amicarbazone, including its metabolites and degradates, in or on the commodities in the table below when present therein as a result of application of amicarbazone to the growing crops in paragraph (a) of this section.  Compliance with the tolerance levels specified below is to be determined by measuring only the sum of amicarbazone [4-amino-N-(1,1-dimethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide]  and its metabolites desamino amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide] and isopropyl-2-hydroxy amicarbazone [N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-hydroxy-1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide], calculated as stoichiometric equivalents of amicarbazone, in or on the commodity. 

2.3	Label Recommendations

2.3.1	Recommendations from Occupational Assessment

The Registration Division should ensure the final label does not include the potential for application to Christmas trees using a mechanically pressurized handgun sprayer.

2.3.2	Recommendations from Residential Assessment

No recommendations are needed based on HED's residential exposure and risk assessment.

3.0	Introduction

3.1	Chemical Identity

Table 3.1  Amicarbazone Nomenclature
Chemical Structure

Empirical Formula
C10H19N5O2
Common Name
Amicarbazone
Company experimental name
MKH 3586
IUPAC name
4-amino-N-tert-butyl-4,5-dihydro-3-isopropyl-5-oxo-1H-1,2,4-triazole-1-carboxamide
CAS Name
4-amino-N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide
CAS Registry Number
129909-90-6
End-use product/EP
66330-46 [Amicarbazone DF Herbicide, 70% ai]
Chemical Class
triazolinone or triazolone (referred to in this document as triazolinone) herbicide
Known Impurities of Concern
N/A

3.2	Physical/Chemical Characteristics

AMICARBAZONE HAS A LOW VAPOR PRESSURE (2.3 x10[-][8] mm Hg at 25 [o]C) and is soluble in water (solubility 4,600 ppm at 20 [o]C).  Based on submitted environmental fate studies, the chemical is moderately persistent in aerobic soil (t1/2= 87 days determined for only one soil, shorter  half-lives were determined under field conditions: t1/2= 19 to 29 days).  It appears to degrade slowly into a number of degradation products under different environmental conditions.  Amicarbazone is stable to direct photolysis (indirect photolysis in natural pond water: t1/2≈73 days); hydrolysis (except at pH 9: t1/2= 66 days); and anaerobic aquatic metabolism (t1/2= >4 years in the solid phase and 533 days in the water phase).  With the exception of natural clear water bodies subjected to sunlight, the major route of dissipation of amicarbazone appears to be bio-transformation.  In clear water bodies subjected to sunlight, indirect photolysis may contribute to amicarbazone dissipation. Although photolysis might occur on soil surfaces (t1/2= 54 days), label-recommended incorporation of the pesticide just after application may render this process unimportant.  A table of physical and chemical properties for amicarbazone is included in Appendix B.

3.3	Pesticide Use Pattern

Arysta LifeSciences North America, LLC is currently seeking use of amicarbazone for control of broadleaf weeds in turf (i.e., lawns, sod farms, golf courses, recreational fields, etc.), ornamentals, non-crop areas, Christmas tree farms and conifers in nurseries.  Amicarbazone is formulated as a 70% dry flowable powder.  It is intended for pre-emergence and post-emergence applications.  Broadcast applications are to be made with ground equipment only; spot treatments are to be made using backpacks and hand held equipment (low pressure, high pressure and trigger sprayer).  The product is not to be applied by aerial equipment or through irrigation systems.

THe herbicide works best when applied and subsequently moved into the soil by watering in or rainfall.  The personal protective equipment (PPE) for the proposed label consists of baseline clothing (i.e., long-sleeved shirt and long pants, shoes and socks) and use of chemical resistant gloves.

Table 3.3.  Summary of Proposed Use Directions for Amicarbazone.
                       Applic. Timing, Type, and  Equip.
                                 Formulation 
                                (EPA Reg. No.)
                           Max. Single Applic. Rate 
                                   (lb ai/A)
                          Max. No. Applic. per Season
                     Max. Seasonal Applic. Rate (lb ai/A)
                                  PHI (days)
                        Use Directions and Limitations
Turf [Golf Course/Sod Farms/Parks/Recreation Areas/Athletic Fields], Landscape plants and Tree Nurseries and Christmas trees farms
Directed,
Broadcast using ground equipment only.

Spot treatment using backpack and compression sprayers.
Amicarbazone DF Herbicide 

(DINAMIC
70 WDG HERBICIDE)*
70% ai

(EPA Reg. No. 66330-46)
0.46 lb ai/A 

(10.5 oz prod/A)

(0.023 lb ai/gal)
1 appl. at the max rate.

2-4 appls. for turf grass at a lower rate.

0.46 lbs ai/ A/yr
N/A
12-hr REI
Application intervals from 14-21 days, but may vary depending on application rate.

Do not apply within 4 weeks of cutting or lifting sod.

Do not apply using aerial equipment or any irrigation system.
[†]  Max Single Application Rate = (70% ai) (1 lbsolid/16 oz) (10.5 oz/A) = 0.46 lb ai/A (1 A/20gal) = 0.023 lb ai/gal.
[‡]  Minimum Spray Volume = 20 gal/A.
* DINAMIC WDG HERBICIDE is an alternate brand name and is the same product as Amicarbazone DF Herbicide.
Note:  The proposed label states that the maximum single application rate for turf is 10 oz. product/A.  However, for purposes of this assessment HED used the maximum single application rate of 10.5 oz. product/A based on information provided by the registrant (MRID No. 48237522), which corresponds to the application rate listed for all crops on the general instructions section of the proposed label. 

3.4	Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the proposed new use of the herbicide amicarbazone on turf, on ornamentals and Christmas trees in nurseries, and in Christmas tree farms.  Amicarbazone is currently registered for use on field corn, and there are tolerances for residues in field corn, in livestock commodities, and in various rotational crops.  Therefore, humans may be exposed to amicarbazone in food and drinking water, since the chemical may be applied directly to growing crops and may reach surface and ground water sources of drinking water.  The currently proposed use is the first residential use of amicarbazone so there is likely to be exposure in residential and non-occupational settings.  While the product does not appear to be intended for homeowner use, there is the potential for homeowners to purchase and apply the product to their own lawns, and the potential for this exposure has been considered in the risk assessment.  In an occupational setting, applicators may be exposed while handling the pesticide prior to application (i.e., mixing/loading), as well as during application.  There is a potential for postapplication exposure for workers re-entering treated fields, but since there is no dermal endpoint (toxicity) associated with amicarbazone, these exposures were not assessed.

Risk assessments have been previously prepared for the existing use of amicarbazone on corn.  This risk assessment considers all exposure pathways based on the existing and proposed new uses of amicarbazone.

3.5	Consideration of Environmental Justice

Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to Address Environmental Justice in Minority Populations and Low-Income Populations," (http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf.  As a part of every pesticide risk assessment, 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 consumption, and activities in and around the home that involve pesticide use in a residential setting.  Extensive data on food consumption patterns are compiled by the USDA under the Continuing Survey of Food Intake 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 based on age, season of the year, ethnic group, and region of the country.  Additionally, OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments are performed when conditions or circumstances warrant.  Whenever appropriate, non-dietary exposures based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or playing on treated areas postapplication are evaluated.  Further considerations are currently in development as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0	Hazard Characterization and Dose-Response Assessment

4.1	Toxicology Studies Available for Analysis

The toxicology database is considered complete for required toxicity studies, and is sufficient for characterizing the human health hazard of amicarbazone.  The database includes oral and dermal toxicity studies, and both routes are consistent with potential exposure scenarios.  Inhalation toxicity data are not available at this time, although it is a possible route of residential and occupational exposure.  HED is developing criteria for determining when the inhalation toxicity study will be required, and may require a study for amicarbazone in the future.  Toxicology studies available for consideration included subchronic oral toxicity in the rat and dog, subchronic (21-day) dermal toxicity in the rat, chronic toxicity in the dog and rat, carcinogenicity in the rat and mouse, developmental toxicity in the rat and rabbit, reproductive toxicity in the rat, acute and subchronic neurotoxicity in the rat, developmental neurotoxicity in the rat, genotoxicity in multiple systems, immunotoxicity in the rat and metabolism in the rat.  Several supplemental nonguideline mechanistic studies were also submitted examining the effects of amicarbazone on thyroid hormones in the rat and neurotoxicity in the rat and mouse.  In addition, a rat metabolism study on the soil metabolite 4-methyl MKH 3586 and bacterial reverse gene mutation assays on oxadiazolinon and triazolinon, two intermediates of amicarbazone, were submitted.  A screening-level search did not identify relevant studies in the published literature.

4.2	Absorption, Distribution, Metabolism, & Elimination (ADME)

Amicarbazone was well-absorbed in the male rat following oral dosing.  Approximately 95% of the administered radioactive dose was recovered within 72 hours after dosing, with the majority (64%) excreted within 24 hours via the urine, and fecal excretion accounting for 27%.  At 72 hours the tissues and carcass contained <1% of the administered dose, with the highest concentrations remaining in the liver, kidney and blood.  Plasma levels of radioactivity were not determined in this study.  Amicarbazone was extensively metabolized, with only 2% and <1% of the administered dose excreted as unchanged parent in the urine and feces, respectively.  The primary routes of metabolism were glucuronidation with excretion in the feces, and deamination and hydroxylation with excretion in the urine.  The major metabolites that were identified were iPR-2-OH DA MKH 3586, a hydroxylated, deaminated product of the parent compound excreted primarily in the urine (32% out of 34% total excretion), tBu-OH DA MKH 3586 (11% in urine and 4% in feces), and iPR-1,2-diOH DA MKH 3586 (6% in urine, <1% in feces).  A glucuronidated metabolite, MKH 3586GA, was excreted primarily in the feces (10% out of 11% total excretion).  Five minor identified metabolites (<3% of dose) and seven unidentified metabolites (all <1% of dose) were isolated in the urine and the feces.

4.2.1	Dermal Absorption

Dermal absorption data were not required for amicarbazone and were not submitted.  An acceptable 21-day dermal toxicity study in the rat was available.  There was no evidence of either systemic toxicity or local dermal irritation.  Using a ratio of the LOAELs from several oral studies in the rat (e.g., subchronic oral, 67 mg/kg/day; subchronic neurotoxicity 33.4 mg/kg/day; reproductive toxicity 33.9 mg/kg/day) to the NOAEL from the 21-day dermal rat study (1000 mg/kg/day), a conservative dermal absorption factor of less than 5% was estimated.  Therefore, dermal absorption is not considered to be a major route of uptake for this chemical and the dermal route of exposure was not considered for this risk assessment.

4.3	Toxicological Effects

Amicarbazone is a triazolinone herbicide which controls broadleaf weeds by selective inhibition of acetolactate synthase, an enzyme involved in photosystem II in plants.  The available database indicates that the major effects in mammals are general toxicity, as evidenced by decreased body weight and weight gain, and liver toxicity.  Decreased body weight and weight gain were observed in studies with rats, mice and rabbits and were the most sensitive toxicological endpoints observed in many of the studies.  In the chronic dog and subchronic rat and dog studies, liver effects were seen, including increased absolute and relative liver weights, slight liver hypertrophy, dilation of sinusoids and increased liver enzymes and cholesterol/triglycerides.

Slight effects on the thyroid gland were also seen in the subchronic rat and dog studies.   These effects included such changes as increased thyroid weight (dog), increased levels of T3 and T4 (rat), hyperplasia (rat) and vacuolization (dog).  However, these thyroid effects were not observed in the chronic rat and dog studies.  Mechanistic studies in rats and in vitro thyroid enzyme assays were performed to further characterize effects on the thyroid.  The mechanistic studies in rats did not show treatment related effects on thyroid weight or microscopic changes in the thyroid at any dose.  Thyroid to blood ratios of [125]I in treated groups were comparable to those of negative controls, indicating the increase in thyroid hormones is most likely not due to increased synthesis; thus, differences in thyroid hormones may be due to metabolism at another site.  In vitro assays indicated that amicarbazone did not affect the iodide organification step in thyroid hormone synthesis or the peripheral metabolism of hormones via deiodinases.  Based on these findings, the liver is implicated as the extra-thyroidal site based on increased liver weights and UDP-glucuronosyltransferase activity.

There was no evidence of increased quantitative or qualitative susceptibility in offspring in the available developmental and reproductive toxicity studies.  In developmental studies with rats and rabbits dosed by gavage, maternal toxicity was observed in the form of decreased body weight (rats), body weight gain (rats and rabbits), and food consumption (rats).  The developmental LOAEL in the rat study was based on multiple skeletal developmental retardations (incomplete ossification/unossification was observed in parietal bones, interparietal bones, supraoccipital bones, squamosal bones, zygoma, pubis, xiphoid, and fontanelle).  However, delayed ossification in the absence of decreased fetal viability (as observed in the 2-generation rat study), is generally not considered to be more severe or qualitatively more significant than decreased body weight and body weight gain in maternal animals.   In the developmental rabbit study, maternal animals were more sensitive than the developing fetuses to amicarbazone.  Decreased body weight gain was seen in maternal animals at the mid dose, while decreased body weights and increased incidences of incomplete ossification were seen in fetuses at the high dose. Maternal effects at the high dose were decreased body weight gain and abortion. In the rat 2-generation reproduction study, both maternal and developmental endpoints were based on decreased body weight and body weight gain.

Neurotoxic effects were seen in an acute oral (gavage) neurotoxicity study in rats.  The clinical signs included eyelid ptosis, decreased approach response in both sexes, and red nasal staining in males.  Two other gavage studies also showed effects of neurotoxicity.  In a nonguideline study looking at the central nervous system (CNS) response in mice, a single oral dose (100 mg/kg) caused minimal functional impairment in males.  This impairment was characterized by increased reaction times to nociceptive stimuli, decreased traction force, impaired motor coordination, sedation, partial ptosis, and mild anticonvulsive effects. In another nonguideline behavioral study in rats, clinical signs (sedation, ptosis, salivation) were observed after a single oral dose.  Additionally, at a high dose, piloerection, Straub phenomenon, and prone position were observed.  Effects were observed at 30 minutes post dose with apparent recovery by 150 minutes post dose; the higher dose groups showed greater persistence of effects.  A dose- and time-dependent effect was demonstrated on motor activity, decreased travel distance, increased resting time, and decreased rearing.  Maternal clonic convulsions were observed in the developmental rat study at 300 mg/kg/day.  In contrast, the subchronic and developmental neurotoxicity studies in rats (both dietary studies) did not result in any signs of neurotoxicity up to 100 mg/kg/day.  

There was no evidence of immunotoxicity in the rat or mouse subchronic and chronic studies. However, in an immunotoxicity study in the female rat, suppression of the humoral response (reduced spleen cells/spleen and suppressed SRBC antibody response), along with reduced absolute spleen weight and decreased body weight/weight gain, were reported at the highest dose tested.  In the dog chronic study, thymic atrophy was observed but only at the highest dose tested.  Immunotoxicity parameters examined in this study (flow cytometer cell phenotyping and immunoglobulin determination) were unaffected by treatment.  In the subchronic dog study, lymphoid hyperplasia of the gall bladder observed at the LOAEL was considered to be a secondary effect to toxicity of the gall bladder and was not observed in the chronic dog study.  

There were no systemic effects observed in the 21-day dermal toxicity study in rats exposed to amicarbazone.  No treatment related signs of dermal irritation were observed.

Both the rat and mouse carcinogenicity studies indicate that amicarbazone is not likely to be carcinogenic to humans.  At the doses tested there was no treatment related increase in tumor incidence when compared to controls.  Dosing in rats and in mice was considered adequate based on body weight and body weight changes in both sexes.  The mutagenicity battery for this chemical is complete and indicates that amicarbazone is not a mutagen.

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

HED has recommended that the 10x FQPA factor be reduced to 1x.  No evidence of increased quantitative or qualitative susceptibility was observed in the rat and rabbit developmental toxicity, rat reproductive toxicity or the rat developmental neurotoxicity studies.  Although neurotoxicity was observed with exposure to amicarbazone in adult animals in gavage studies, no neurotoxic effects were observed in offspring in the rat developmental neurotoxicity study at dose levels that resulted in general systemic toxicity (decreased offspring and maternal body weight).  Potential immunotoxic effects (chronic dog and rat immunotoxicity studies) were observed only at doses greater than those selected for risk assessment purposes and are therefore not of concern.

Although the conditionally required subchronic (21/28 study) inhalation study is not available, the most sensitive toxicity endpoint identified in the oral studies was used for risk assessment.  In addition, conservative health protective assumptions were made in the exposure assessment.   Therefore, HED did not retain a database uncertainty factor to account for absence of the study. Given the overall completeness of the database, there are no database uncertainty factors to be retained during risk assessment, and therefore the LOC is an MOE of 100, based on the combined uncertainty factors of 100X (10X for interspecies extrapolation and 10X for intraspecies variability).  MOEs greater than the LOC of 100 are not of concern.

4.4.1	Completeness of the Toxicology Database

The toxicology database is complete for amicarbazone.  The scientific quality of the database is adequate and the toxicity profile can be characterized for a wide array of effects, including potential developmental and reproductive toxicity, immunotoxicity and neurotoxicity.  Developmental toxicity studies in the rat and rabbit, a rat reproductive toxicity study and acute, subchronic and developmental neurotoxicity studies have been submitted.  An immunotoxicity study in the rat has been submitted since the previous risk assessment, and is considered to be acceptable.

The Agency is currently in the process of evaluating its guidance for inhalation toxicity data requirements; the need for an inhalation study for amicarbazone will be determined once the guidance has been finalized.  Although subchronic inhalation data on amicarbazone are not available and an oral study was selected for inhalation risk assessment, the selected points of departure are considered adequately protective for all exposed populations.  The acute inhalation toxicity of amicarbazone is low (Category IV), the vapor pressure is low, and inhalation MOEs calculated for residential handler exposure using the oral study are high (3,600 to 220,000).  Therefore, an additional 10x database uncertainty factor was not retained for lack of inhalation toxicity data.

4.4.2	Evidence of Neurotoxicity

Neurotoxicity was observed following exposure to amicarbazone in gavage dosing studies.  In the rat acute neurotoxicity study, eyelid ptosis and decreased approach response in males and females, along with red nasal stain in males, were observed at 3 hrs post-dosing at the LOAEL of 20 mg/kg.  Decreased total locomotor activity was observed at higher dose levels, along with other effects such as tremors, impaired righting reflex, rigid muscle tone and repetitive forepaw movements.  Mortality was reported at the highest dose tested (400 mg/kg/day).  Effects did not persist except for nasal/oral staining.  Maternal clonic convulsions were observed in 3/27 rats in the developmental rat study following exposure to 300 mg/kg/day amicarbazone.  Nonguideline behavioral, single gavage dose studies were conducted in the rat and the mouse.  Transient sedation, ptosis and salivation were reported at 10 mg/kg in the rat in only 1-2 animals but these effects were more pronounced at 20 mg/kg, and minimal CNS functional impairment in mice (increased response times to nociceptive stimulus, reduced traction force and impaired motor coordination, sedation and partial ptosis) was observed at 100 mg/kg.  

In the rat subchronic and developmental neurotoxicity studies, both using dietary exposure, no evidence of neurotoxicity was observed.  This risk assessment is therefore protective of potential neurotoxicity, since the acute dietary assessment was based on the NOAEL of 10 mg/kg from the acute neurotoxicity study; other endpoints for exposure were based on lower NOAELs.

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

There was no evidence of increased qualitative or quantitative susceptibility in fetuses of the rat or rabbit developmental toxicity studies or in offspring of the rat reproductive toxicity or developmental neurotoxicity studies.  Fetal and offspring effects in these studies, which included decreases in body weight and delays in ossification, were observed at maternally toxic dose levels and were not considered evidence of increased severity relative to the adult animal.

Thyroid effects were observed in the rat and dog studies.  Thyroid histopathology was also examined in the rat developmental neurotoxicity study; no effects on the thyroid were observed at any dose tested.  Effects on body weight or neurobehavioral toxicity were not reported in offspring at doses lower than those causing maternal effects.  Therefore, there is no evidence of increased susceptibility for thyroid effects in the developing or young animal and the points of departure selected for risk assessment are considered protective.

4.4.4	Residual Uncertainty in the Exposure Database

There is no residual uncertainty with respect to the exposure assessments conducted for amicarbazone.  The dietary exposure estimates incorporate conservative estimates of all residues of concern from both the food and drinking water exposure pathways.  The aggregate assessment including exposure from the proposed use on turf also used high-end default assumptions; the potential for children's exposure to residues on treated turf was based on high-end turf residues expected on the day of application, even though the label indicates the greatest herbicidal effectiveness is achieved through immediate watering or rainfall.

4.5	Toxicity Endpoint and Point of Departure Selections

4.5.1	Dose-Response Assessment

The detailed summaries of the toxicity studies used for selecting toxicity endpoints and points of departure for various exposure scenarios are presented in Appendix A.4 of this assessment.

For acute dietary exposure assessment (all populations), studies considered were the rat acute oral neurotoxicity study and developmental toxicity studies in the rat and rabbit.  The NOAEL of 10 mg/kg from the acute neurotoxicity study, based on eyelid ptosis and diminished approach response observed at the LOAEL of 20 mg/kg, was selected for all populations.  These effects showed a dose-response, and additional findings were observed at higher doses, including mortality at 400 mg/kg.  The selected NOAEL is protective of potential toxicity to offspring and young children.  Findings observed in the developmental toxicity and developmental neurotoxicity studies (delays in ossification, decreased body weight) were not considered to be single-dose effects.

For chronic dietary exposure, the rat dietary chronic toxicity/carcinogenicity and the dog dietary chronic toxicity studies were considered and were selected as co-critical studies.  Both identified a NOAEL of 2.3 mg/kg/day.  In the rat, effects observed at the LOAEL of 25.3 mg/kg/day were decreased body weight and weight gain.  In the dog, effects observed at the LOAEL of 8.7 mg/kg/day were increased absolute and relative liver weights and O-demethylase in males; increased globulin and cytochrome p450 in females; and increased triglycerides and cholesterol in both sexes.

For incidental oral exposure of short-term (1-30 days) duration, studies considered were the subchronic oral toxicity studies in the rat and dog, subchronic oral neurotoxicity and developmental neurotoxicity studies in the rat, reproductive toxicity in the rat and developmental toxicity studies in the rat and rabbit.  The dog subchronic oral toxicity study was selected, with a NOAEL of 6.28 mg/kg/day.  At the LOAEL of 25 mg/kg/day, numerous effects were observed.  These included increased thyroid vacuolization, decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase and O-demethylase in both sexes.  The point of departure in this study was supported by a similar offspring NOAEL in the rat reproductive toxicity study (6.4 mg/kg/day, with a LOAEL of 33.9 mg/kg/day) and maternal NOAEL in the rabbit developmental toxicity study (5 mg/kg/day, with a LOAEL of 20 mg/kg/day), both based on decreased body weight/weight gain, and NOAEL of 7.8 mg/kg/day in the subchronic rat neurotoxicity study, based on decreased body weight and body weight gain in females at 38.2 mg/kg/day.  The slightly higher NOAEL of the dog subchronic study compared to the reproductive and developmental studies was likely due to dose spacing.  All other NOAELs identified in the database for studies of the appropriate duration were higher.

No dermal endpoint was selected for risk assessment, based on the lack of systemic or local dermal toxicity in the rat dermal exposure study.  Comparison of rat oral study LOAELs to the dermal NOAEL supported a low level of dermal absorption (see Section 4.2.1), and no effects of specific concern for the developing young were identified; therefore, the dermal risk assessment was not required.

For inhalation exposure of short- or intermediate-term duration, the studies listed above for incidental oral exposure were considered; inhalation data were not available.  The dog subchronic oral toxicity study was selected for the reasons previously discussed.

Based on the current and proposed use patterns, long-term dermal and inhalation exposure are not expected, either in residential or occupational settings; therefore, no endpoints were selected for risk assessment for long-term durations of exposure.

4.5.2	Recommendation for Combining Routes of Exposures for Risk Assessment

Dermal exposure was not assessed due to a lack of toxicity via the dermal route.  Since adults were assessed for potential inhalation exposure while applying amicarbazone to lawns, this exposure should be combined with background levels in food and water to determine aggregate exposure.  For children's residential exposure, only oral postapplication exposure was assessed, and this exposure should be combined with exposure from food and drinking water to determine aggregate exposure.  For occupational workers, only inhalation exposure and risk were assessed.

4.5.3	Cancer Classification and Risk Assessment Recommendation

Under the revised 2005 Agency cancer assessment guidelines, amicarbazone is classified as "not likely to be a human carcinogen."  There were no treatment-related increases in the incidence of tumors in either the rat or mouse carcinogenicity studies, both of which were tested at adequate doses.

4.5.4	Summary of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment

Table 4.5.4.1  Summary of Toxicological Doses and Endpoints for Amicarbazone for Use in Dietary and Non-Occupational Human Health Risk Assessments
                              Exposure/ Scenario
                              Point of Departure
                        Uncertainty/FQPA Safety Factors
                RfD, PAD, Level of Concern for Risk Assessment
                        Study and Toxicological Effects
Acute Dietary (General Population, including Infants and Children and Females 13-49 years of age)
NOAEL= 10 mg/kg/day
UFA= 10x
UFH=10x
FQPA SF= 1x

Acute RfD = 0.10 mg/kg/day

aPAD =0.10 mg/kg/day
Acute Oral Neurotoxicity Screening Battery

LOAEL = 20 mg/kg/day based on eyelid ptosis and decreased approach response (both sexes) and red nasal staining in males
Chronic Dietary (General Population, including Infants and Children and Females 13-49 years of age)
NOAEL= 2.3 mg/kg/day
UFA= 10x
UFH=10x
FQPA SF= 1x

Chronic RfD = 0.023
mg/kg/day

cPAD = 0.023 mg/kg/day
Chronic Oral Rat and Chronic Oral Dog (co-critical)

Rat LOAEL = 25.3 mg/kg/day based on decreased body weight and weight gain
Dog LOAEL = 8.7 mg/kg/day based on liver effects, including increased absolute/relative liver weights, triglycerides and cholesterol 
Incidental Oral Short- Term (1-30 days)
NOAEL= 6.28 mg/kg/day
UFA= 10x
UFH=10x
FQPA SF= 1x

Residential LOC for MOE = 100
90-Day Oral Toxicity in Dogs

LOAEL = 25 mg/kg/day based on increased thyroid vacuolization and decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights, and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase and O-demethylase in both sexes.
Dermal (all durations)
No systemic toxicity by the dermal route was seen at the limit dose.  Evidence of low dermal absorption was estimated by comparison of oral and dermal studies in the rat.
Inhalation Short-  and Intermediate-Term (1-30 days and 1-6 months, respectively)
NOAEL= 6.28 mg/kg/day

(inhalation toxicity considered equivalent to oral toxicity)
UFA= 10x
UFH=10x
FQPA SF= 1x

Residential LOC for MOE = 100
90-Day Oral Toxicity in Dogs

LOAEL = 25 mg/kg/day based on increased thyroid vacuolization and decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights, and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase and O-demethylase in both sexes.
Cancer (oral, dermal, inhalation)
Classification:  There was no treatment-related increase in tumor incidence when compared to control.  Dosing was considered adequate.  This chemical is not likely to be a carcinogen.
Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and  used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures.  NOAEL = no observed adverse effect level.  LOAEL = lowest observed adverse effect level.  UF = uncertainty factor.  UFA = extrapolation from animal to human (interspecies).  UFH = potential variation in sensitivity among members of the human population (intraspecies).  FQPA SF = FQPA Safety Factor.  RfD = reference dose.  MOE = margin of exposure.  LOC = level of concern.  N/A = not applicable.

Table 4.5.4.2 Summary of Toxicological Doses and Endpoints for Amicarbazone for Use in Occupational Human Health Risk Assessments
                              Exposure/ Scenario
                              Point of Departure
                              Uncertainty Factors
                     Level of Concern for Risk Assessment
                        Study and Toxicological Effects
Dermal (all durations)
No systemic toxicity by the dermal route was seen at the limit dose.  Evidence of low dermal absorption was estimated by comparison of oral and dermal studies in the rat.
Inhalation Short- and Intermediate-Term (1-30 days and 1-6 months, respectively)
NOAEL= 6.28 mg/kg/day

(inhalation toxicity considered equivalent to oral toxicity)
UFA=10x
UFH=10x
Occupational LOC for MOE = 100
90-Day Oral Toxicity in Dogs

LOAEL = 25 mg/kg/day based on increased thyroid vacuolization and decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights, and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase and O-demethylase in both sexes.
Cancer (oral, dermal, inhalation)
Classification:  There was no treatment-related increase in tumor incidence when compared to control.  Dosing was considered adequate.  This chemical is not likely to be a carcinogen.
Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and  used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures.  NOAEL = no observed adverse effect level.  LOAEL = lowest observed adverse effect level.  UF = uncertainty factor.  UFA = extrapolation from animal to human (interspecies).  UFH = potential variation in sensitivity among members of the human population (intraspecies).  MOE = margin of exposure.  LOC = level of concern.  N/A = not applicable.

5.0	Dietary Exposure and Risk Assessment 

5.1	Metabolite/Degradate Residue Profile

5.1.1	Summary of Plant and Animal Metabolism Studies

There have been no new data submitted with respect to metabolism in plants and livestock.  HED previously concluded that the nature of the residue is adequately understood based on studies in corn, in rotational crops and in livestock.

Based on the results of the corn metabolism study, the proposed major metabolic pathway in corn involves the deamination of the triazole amino group to form the desamino metabolite (DA amicarbazone) followed by hydroxylation at the tertiary carbon of the isopropyl group to form iPr-2-OH desamino amicarbazone.  Additional pathways involve hydroxylation of the isopropyl methyl to form iPr-1-OH desamino amicarbazone, followed by glucosidation; hydroxylation of the t-butyl and isopropyl groups to form tBu-iPr-2-diOH desamion amicarbazone. In addition, desamino amicarbazone formed an N-glucoside; and glucosidation of hydroxylated desamino amicarbazone formed several minor O-glucosides. 

The proposed mechanism of metabolism in livestock is loss of the triazole amino group, yielding desamino amicarbazone, followed by hydroxylation of the isopropyl methine carbon to form iPr-2-OH desamino amicarbazone.  Dehydration would then result in iPr-Ene desamino amicarbazone, which may undergo epoxidation and hydrolysis to form iPr-1,2-diOH desamino amicarbazone.  Hydroxylation of desamino amicarbazone at an isopropyl methyl carbon would yield iPr-1-OH desamino amicarbazone, which may then undergo oxidation to form iPr-Acid desamino amicarbazone.  Minor pathways involve hydroxylation of the t-butyl group of amicarbazone to form tBu-OH amicarbazone, and hydrolytic cleavage of the carboxamide side chain of amicarbazone or desamino amicarbazone to form triazolinone amicarbazone or triazolinone desamino amicarbazone.

Existing data indicate that uptake by rotational crops is likely to occur.  The metabolites identified in the rotational crops were similar to those found in the primary crop corn metabolism study.  The total level of the glucose conjugates was higher in rotated wheat commodities than in corn commodities.  Two minor metabolites identified in the rotational crops, triazolinone amicarbazone and N-Me desamino amicarbazone, were not found in the corn metabolism study.

5.1.2	Summary of Environmental Degradation

Amicarbazone is soluble in water (solubility 4,600 ppm at 20 [o]C).  Based on submitted environmental fate studies, the chemical is moderately persistent in aerobic soil.  It appears to degrade slowly into a number of degradation products under different environmental conditions.  Amicarbazone is stable to direct photolysis, hydrolysis, and anaerobic aquatic metabolism.  With the exception of natural clear water bodies subjected to sunlight, the major route of dissipation of amicarbazone appears to be bio-transformation.  In clear water bodies subjected to sunlight, indirect photolysis may contribute to amicarbazone dissipation. Although photolysis might occur on soil surfaces (t1/2= 54 days), label-recommended incorporation of the pesticide just after application may render this process unimportant. 

Laboratory and field studies identified only three major degradates for amicarbazone: desamino (DA amicarbazone), N-methyl desamino and decarboxamide.  Desamino and N-methyl desamino were both the result of biodegradation in aerobic soil in the laboratory and in the field, while desamino alone also resulted from indirect photolysis in aqueous systems.  Decarboxamide resulted only from hydrolysis in alkaline aqueous systems (29% of applied dose after 30 hours at pH 9).  Mobility studies were the only submitted studies for the major three degradates of amicarbazone; however, data from the single aerobic soil study on the parent suggests that both desamino and N-methyl desamino are highly persistent (did not show decline within a period of a year).  

Available data suggest that amicarbazone is very highly mobile (Koc range= 16.7 to 37.0 L kg[-1], from adsorption/desorption and aged leaching studies).  Additionally, adsorption/desorption and aged leaching studies suggest that all of the three major degradates of amicarbazone are very highly mobile (ASTM 1996).  Determined Koc values were: desamino (Koc range= 26.4 to 42.3 L kg[-1]), N-methyl desamino (Koc range= 34.3 to 56.4 L kg[-1]) and decarboxamide (Koc range= 9.4 to 16.2 L kg[-1]). 

Given the moderate persistence/high mobility and solubility of the parent compound and the apparent high persistence/high mobility of its two degradates, amicarbazone is expected to dissipate slowly and at the same time be vulnerable to leaching/run-off.  The desamino and N-methyl desamino degradates are also expected to behave similarly to the parent and become the major terminal degradates of this chemical in surface and ground water.

When amicarbazone is transported into alkaline ground water, surface water bodies and/or soils, the third degradate (decarboxamide) is expected to become an important contaminant of surface and/or ground water for two reasons: it is the major hydrolysis degradate of parent in alkaline conditions and is highly mobile.  The potential contamination of surface and ground water with this degradate is expected to be correlated to its persistence.  Unfortunately, the only studies submitted on the degradates were adsorption/desorption, so that the persistence of decarboxamide is unknown.

However, since the amicarbazone is not to be applied on soils with pH higher than 7.4, only the fate of the chemical in neutral and acidic conditions is relevant for the current assessment.

5.1.3	Comparison of Metabolic Pathways

In an acceptable rat metabolism study using amicarbazone, 95% of the radioactive dose was recovered within 72 hours post dosing.  Urinary excretion accounted for 64% of the dose, indicating substantial absorption, and fecal excretion accounted for 27% of the dose within 24 hours of dosing.  The parent compound only accounted for approximately 3% of the radioactive dose, indicating rapid metabolism.  Three major metabolites were identified in the excreta, iPr-2-OH desamino MKH, tBu-OH desamino MKH, and glucuronic acid conjugates.  Based on the metabolic profile, the metabolism of amicarbazone in rats primarily involves deamination followed by hydroxylation with elimination in the urine.  The parent also undergoes glucuronic acid conjugation and elimination in the feces.

A second rat metabolism study was performed with an environmental degradate of amicarbazone, 4-methyl amicarbazone.  The routes of excretion for this compound are similar to that of the parent.  Over 90% of the radioactive dose was eliminated within 96 hours, the majority eliminated through the urine and feces within 24 hours of administration.  Hydroxylation of the soil metabolite at the isopropyl moiety formed the major metabolite, 4-Me-i-Pr-2-OH desamino amicarbazone, which was found primarily in urine.  Further hydroxylation at the tertiary butyl group resulted in the formation of 4-Me-t-Bu-iPr-2-di-OH desamino amicarbazone.  Alternatively, additional hydroxylation of the isopropyl moiety formed 4-Me-iPr-1,2-di-OH desamino amicarbazone.  The pathway for the metabolism of 4-methyl desamino amicarbazone in rats primarily involves a series of hydroxylation reactions.  No conjugates were detected.    

Comparing studies in different species, the major plant and livestock metabolites desamino amicarbazone and iPr-2-OH desamino amicarbazone are also observed in the rat.  Desamino amicarbazone is also a major environmental degradate.  However, the other significant environmental degradate, N-methyl-desamino amicarbazone, is not seen as a rat metabolite.

5.1.4	Residues of Concern Summary and Rationale

No changes have been made to the previous recommendations with respect to parent amicarbazone and metabolites with respect to tolerance enforcement and risk assessment.  In general, metabolites were included in risk assessment based on the presence of the triazolinone ring, and potential toxicity equivalent to that of the parent.  Metabolites were recommended for inclusion in the tolerance expression based on the potential for toxicity similar to that of the parent, as well as the potential for significant levels in crops and livestock, as well as the metabolites/degradates determined by the enforcement methods.

The residues of concern in field corn consist of the parent amicarbazone, the desamino metabolite and iPr-2-OH desamino amicarbazone.  Based on the triazolinone ring still being present, HED cannot rule out these metabolites as possessing similar toxicity as parent amicarbazone.  They are also major metabolites (> 10% TRR) in at least two corn matrices (grain, fodder) and are determined along with the parent by the tolerance enforcement method.  Therefore, HED concluded that these metabolites should be included along with the parent as residues in the field corn tolerances and dietary risk assessment.

For livestock commodities the same residues as above are to be included in tolerances for enforcement purposes.  The livestock enforcement method measures all three compounds as iPr-2-OH desamino amicarbazone by oxidizing the parent and desamino amicarbazone with potassium permanganate.  Each of the three compounds is also a major residue in multiple matrices across the ruminant and poultry metabolism studies.  For risk assessment purposes, HED concluded that additional metabolites needed to be included with the three residues to be used for tolerance enforcement.  In ruminants, the metabolite tBuOH amicarbazone was included based on its significant levels in milk, muscle and kidney.  For poultry HED recommended that the metabolites iPr-Acid desamino amicarbazone, iPr-1-OH desamino amicarbazone, and iPr-Ene desamino amicarbazone be considered in the risk assessment due to their presence as major residues in liver, fat, or eggs.  These additional metabolites to be included in the risk assessment all contain the triazolinone ring.

In rotational crops the residues for tolerance enforcement are the same as those in the primary crop field corn: parent and the metabolites desamino amicarbazone and iPr-2-OH desamino amicarbazone.  These compounds are determined by the plant enforcement method and were major residues in many of the samples analyzed in the confined study.  In addition, quantifiable levels of these residues were observed in all matrices except turnip roots in the field rotational crop studies.  For risk assessment purposes in rotational crops, the same three residues should be considered.  In addition, for assessing human dietary exposure to residues in wheat grain (or any other rotated cereal grains), the two glucose conjugates of desamino OH amicarbazone should be included.  These conjugates constituted about 30% of the grain TRR at the 138- and 364-day plant back intervals.  Although these two conjugates were also major residues in the feed items wheat forage, hay and straw, the combined levels of the parent, desamino amicarbazone, and iPr-2-OH desamino amicarbazone exceed the total levels of the conjugates in forage and hay, the feed items contributing the most to the livestock dietary burdens.  Therefore, HED concluded that the use of the parent, desamino amicarbazone, and iPr-2-OH desamino amicarbazone provides a reasonable estimate of livestock dietary burden, and the conjugates do not need to be considered in the livestock component of the exposure assessment.		

HED previously determined that the residues of concern in drinking water for dietary risk assessment are the parent amicarbazone and the desamino (DA amicarbazone) and N-methyl-desamino degradates, based on the rationale provided below.

Amicarbazone is highly soluble in water, moderately persistent in aerobic soil, and highly mobile.  Amicarbazone is stable to direct photolysis; hydrolysis (except at pH 9: t1/2= 66 days); and anaerobic aquatic metabolism.  The three major degradates observed in the fate studies are the desamino, N-methyl-desamino, and decarboxamide.  All three of these degradates are very highly mobile.  Data from the single aerobic soil study on the parent suggest that both desamino amicarbazone and N-methyl-desamino are highly persistent.  The decarboxamide was observed only in the alkaline hydrolysis study.  

Given the moderate persistence/high mobility and solubility of the parent and the apparent high persistence/high mobility of its two degradates, parent is expected to dissipate slowly and at the same time be vulnerable to leaching/run-off.  The resultant desamino and N-methyl-desamino degradates are also expected to behave similarly to the parent and become the major terminal residues of this chemical in surface and ground water.  Only in cases where amicarbazone is transported into alkaline ground water, surface water bodies and/or soils, the third degradate (decarboxamide) is expected to become important.  Amicarbazone is not recommended to be applied on calcareous soils with pH values substantially higher than 7.

With respect to the toxicity of the three degradates, HED included them in the risk assessment based on the presence of the triazolinone moiety in all their structures.  Using the above information, the parent plus the desamino and N-methyl-desamino degradates need to be included in the drinking water component of the risk assessment based on the persistence and high mobility of these degradates along with their assumed similar toxicities to the parent.  The decarboxamide does not need to be included in drinking water assessments due to the limited conditions (pH>9) under which it could be a major degradate.

Table 5.1.4  Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression.[1]
Matrix
Residues included in Risk Assessment
Residues included in Tolerance Expression
Plants
Primary Crop
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone 

Rotational Crop
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone 
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone 
Livestock
Ruminant
amicarbazone,
desamino MHK amicarbazone,
iPr-2-OH desamino amicarbazone, tBuOH amicarbazone
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone 

Poultry
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone, iPr-Acid desamino amicarbazone, iPr-1-OH desamino amicarbazone, iPr-Ene desamino amicarbazone
amicarbazone,
desamino amicarbazone,
iPr-2-OH desamino amicarbazone 
Drinking Water
amicarbazone,
desamino amicarbazone,
N-methyl-desamino amicarbazone
Not Applicable
[1] Chemical structures for the residues of concern for tolerance enforcement purposes are provided in Appendix B.

5.2	Food Residue Profile

The previously submitted data were considered adequate to support tolerances for residues in corn and various livestock commodities, as well as for inadvertent residues in rotational crop commodities.  Additional storage stability data in crops were requested as a condition of registration when the first registration on corn was evaluated; some of these data, including storage stability for field corn grain, forage and stover, have been submitted and have been reviewed.  While these data demonstrated that total residues of amicarbazone were not stable in corn forage and stover, the impact of the decline in residues is minimal since a correction factor can be determined from the submitted data.  Furthermore, the decline in these 2 livestock feed items is not expected to have an impact on the residue levels used in the dietary assessment.  Additional storage stability data on mustard greens and turnip roots remain outstanding.  However, no additional information related to residues in food was needed in conjunction with the proposed use on turf, ornamentals and Christmas trees.

5.3	Water Residue Profile
Memo, A. Shelby, 9/7/2011, D384659

The residues of concern in drinking water for risk assessment are parent amicarbazone and its desamino and N-methyl desamino degradates.  The surface water and groundwater estimates were calculated using the PRZM/EXAMS and SCI-GROW Models, respectively.  The estimated drinking water concentrations (EDWCs) given in Table 5.3 are the total toxic residues of amicarbazone, which include the desamino and N-methyl desamino degradates as well as unextracted residues.  The EDWCs represent upper-bound estimates that might be found in surface water and groundwater as a result of the application of amicarbazone on turf and conifers one time at the maximum yearly rate (worst case scenario).  The estimated concentration in groundwater was significantly higher than the surface water estimates.

Table 5.3.	Summary of Estimated Surface Water and Groundwater Concentrations for Amicarbazone.
Scenario
                          Surface Water Conc., ppb a
                           Groundwater Conc., ppb b
Acute
                                     33.3
                                      136
                                       
Chronic (non-cancer)
                                     25.8

Chronic (cancer)
                                     11.1

[a] From the Tier II PRZM-EXAMS - Index Reservoir model.  Input parameters are based on the maximum single application rate.  The aerobic aquatic metabolism half life was assumed to be 2816 days, based on the assumption of 2x the aerobic soil metabolism half life.  The aerobic soil metabolism half life was 1408 days, which was the 90[th] percentile average of 5 total toxic residue half-life estimates ranging from 533 to 1733 days.
[b] From the SCI-GROW model.  Input parameters include the maximum single application rate, and a median aerobic soil metabolism half life of 1155 days.

5.4	Dietary Risk Assessment
Memo, D. Dotson, 10/7/2011, D394925

5.4.1	Description of Residue Data Used in Dietary Assessment

The last dietary exposure assessment performed for amicarbazone was done in 2005, when tolerances were first established for the chemical (D313754, D. Dotson, 4/19/2005).  Residues of concern as described above were included in both the previous and current assessments.

In order to account for the metabolites recommended for inclusion in the risk assessment, in addition to those recommended for tolerance enforcement, HED multiplied the recommended (now established) tolerances by factors to account for the additional metabolites.  These factors ranged from 1.2 (for meat and meat by-products) to 1.8X (wheat commodities and poultry liver) the tolerance levels.

The estimate of potential amicarbazone residues in groundwater (136 ppb) was significantly higher than any of the surface water estimates, and was therefore incorporated into the dietary exposure assessment for "water, direct, all sources" and "water, indirect, all sources."  Although no dietary risks of concern were identified, HED notes that the drinking water exposure is the highest contributor to overall dietary exposure and risk.  For both acute and chronic dietary exposure for all infants, the most highly exposure population subgroup, drinking water constitutes approximately 90% of overall exposure to amicarbazone.  Based on the inclusion of the degradates in addition to the parent, as well as unextracted residues in the assessment, the drinking water concentrations are considered to be upper bound.  Exclusion of degradates and bound residue results in reduction of EDWCs by as much as 70%.  Exclusion of bound residues results in reduction of EDWCs by as much as 20%.  Along with the conservative assumptions used for food residues, the overall dietary exposure assessment is considered to be conservative, and actual exposure to amicarbazone through food and water is likely to be lower.

5.4.2	Percent Crop Treated Used in Dietary Assessment

The acute and chronic dietary exposure assessments based on food alone include the assumption of 100% crop treated (PCT) for the existing use on corn.  

5.4.3	Acute Dietary Risk Assessment

The acute risk estimates are below HED's level of concern for all population subgroups, including those comprised of infants and children.  Generally, HED is concerned when risk estimates exceed 100% of the population adjusted dose (PAD).  The acute risk estimate for the general U.S. population is 8.8% of the acute PAD (aPAD).  The population subgroup with the highest acute dietary risk estimate is All Infants, which uses 29% of the aPAD.  As percent crop treated estimates were not incorporated into the acute assessment, the risk estimates are being reported at the 95[th] percentile of exposure.

5.4.4	Chronic Dietary Risk Assessment

Chronic risk estimates are below HED's level of concern for all population subgroups.  The risk estimate for the general U.S. population is 17% of the chronic PAD (cPAD).  The most highly exposed population subgroup is All Infants (<1 year old), which uses 48% of the cPAD.

5.4.5	Summary Table

 Table 5.4.5.  Summary of Dietary (Food and Drinking Water) Exposure and Risk for Amicarbazone.
                              Population Subgroup
                                 Acute Dietary
                               (95th Percentile)
                                Chronic Dietary
                                     Cancer
                                        
                          Dietary Exposure (mg/kg/day)
                                    % aPAD*
                                Dietary Exposure
                                  (mg/kg/day)
                                    % cPAD*
                                Dietary Exposure
                                  (mg/kg/day)
                                      Risk
 General U.S. Population
0.008807
                                      8.8
0.003889
17
                                      N/A
                                      N/A
 All Infants (< 1 year old)
0.029429
                                      29
0.010987
48
                                        
                                        
 Children 1-2 years old
0.014750
                                      15
0.006921
30
 
 
 Children 3-5 years old
0.013469
                                      13
0.006474
28
 
 
 Children 6-12 years old
0.009356
                                      9.4
0.004469
19
 
 
 Youth 13-19 years old
0.006999
                                      7.0
0.003120
14
 
 
 Adults 20-49 years old
0.007621
                                      7.6
0.003472
15
 
 
 Adults 50+ years old
0.006711
                                      6.7
0.003441
15
 
 
 Females 13-49 years old
0.007603
                                      7.6
0.003431
15
 
 
*Population subgroups with the highest exposure are shown in bold.
6.0 Residential (Non-Occupational) Exposure/Risk Characterization
Memo, Z. Figueroa, 11/30/2011, D394924

6.1	Residential Handler Exposure

The Agency uses the term "Handlers" to describe those individuals who are involved in the pesticide application process.  Based on the proposed label (EPA Reg. No. 66330-46) and information provided by the registrant (MRID No. 48237522), the product is to be applied by professional applicators.  The proposed label includes occupational/commercial mixing/loading activities, PPE and REI requirements.  However, since the proposed label does not identify this product as a restricted use pesticide, a residential handler exposure assessment was performed to be protective of potential homeowner exposure.  

The following exposure scenarios were used to assess residential handlers for broadcast and spot treatments:

   * Mixing/Loading/Applying for Sprays with Backpack Sprayer;
   * Mixing/Loading/Applying for Sprays Using Low-Pressure Handwand; and,
   * Mixing/Loading/Applying for Sprays with Hand-Gun Sprayer (hose-end).

In the absence of chemical-specific data for amicarbazone, HED used standard assumptions with respect to the exposure expected for residential handlers.  These standard unit exposures were from the PHED Surrogate Exposure Guide for Residential Handlers, Version 1.1, August 1998 and the Occupational Residential Exposure Task Force (ORETF) (MRID 44972201). 

Information regarding area treated for the various use scenarios was provided by the Science Advisory Council for Exposure Policy #12: Recommended Revisions to the Standard Operating Procedures (SOPs) for Residential Exposure Assessments.  Only short-term durations of exposure were assessed based on the proposed label, and a standard body weight of 70 kg was assumed for adults applying amicarbazone to turf.

Table 6.1.  Amicarbazone Residential Handler Exposure and Risk for Use on Turf.
                              Exposure Scenario 
                                  Target MOE
                           Inhalation Unit Exposure
                         Maximum Application Rate [a]
                      Amount Treated or Handled Daily [b]
                                  Inhalation
                                       
                                       
                                       
                                       
                                       
                                   Dose [c]
                                    MOE [d]
                            Mixer/Loader/Applicator
Backpack Sprayer
(Ground Directed)
                                      100
                                     0.03
                               [Baseline, PHED]
                                0.023 lb ai/gal
                                     5 gal
                                   0.0000493
                                    130,000
Low Pressure Handwand
                                      100
                                     1.063
                               [Baseline, PHED]
                                       
                                     5 gal
                                    0.00175
                                     3,600
Hose-end (for handgun sprayer)
(Mix your own)
                                      100
                                     0.017
                                  [Baseline]
                                [ORETF-OMA004]
                                0.012 lb ai/gal
                                     5 Gal
                                   0.000028
                                    220,000

                                       
                                       
                                 0.46 lb ai/A
                                     0.5 A
                                   0.000056
                                    110,000
Inhalation Unit Exposures taken from: PHED Surrogate Exposure Guide for Residential Handlers, Version 1.1, August 1998; and Occupational Residential Exposure Task Force (ORETF) Study Data (MRID 449722-01).
a.  Application Rate = (70% ai) (1 lbsolid/16 oz) (10.5 oz/A) = 0.46 lb ai/A (1 A/20gal) = 0.023 lb ai/gal.
b.  Policy 12: Recommended Revision to the SOPs for Residential Exposure Assessment.
c.  Inhalation Dose (mg/kg/day) = Inhalation Unit Exposure (mg/lb ai) * Application Rate * Acres Treated or Amount Handled / Body Weight (70 kg).
d.  Inhalation MOE = NOAEL (6.28 mg/kg/day) / Inhalation Dose (mg/kg/day).

A quantitative dermal assessment for residential handlers was not conducted since no systemic toxicity by the dermal route was seen at the limit dose in the dermal toxicity study.  Handler inhalation exposure scenarios resulted in MOEs significantly greater than 100 and are not of concern.

6.2	Postapplication Exposure

Amicarbazone residential postapplication scenarios include children (3 to 6 years) playing on treated turf, adults performing yard work on treated turf and adults playing golf on treated turf.  
As a result, a wide array of individuals of varying ages can potentially be exposed when they engage in activities in areas that have been treated.

In assessing potential dermal postapplication exposure and risk estimates, HED assumes that pesticide residues are transferred to the skin of adults/children who enter treated yards for recreation or other activities such as yard work and golfing.  However, a quantitative postapplication dermal exposure assessment was not performed for either adults or children since no dermal endpoints were selected.  In addition, a quantitative postapplication inhalation exposure assessment was not conducted, based on the application rate and the low vapor pressure, and because the inhalation assessment for handlers is expected to be protective of potential postapplication exposure and risk.

The postapplication exposures for children playing on treated turf resulting in incidental oral exposure as a result of mouthing behaviors were assessed using the HED Draft Standard Operating Procedures (SOP's) for Residential Exposure Assessments, 2000.  Since no chemical-specific residue data were submitted for this action, postapplication exposures were assessed using default assumptions from the Residential Exposure SOPs.

In order to assess non-dietary ingestion (hand-to-mouth, or HTM) exposure, HED assumes that pesticide residues are transferred to the skin of children playing on treated areas and are subsequently ingested as a result of hand-to-mouth transfer.

Typical assumptions were used in assessing HTM exposure for amicarbazone, including the following:
 
   * On the day of application, it is assumed that 5% of the application rate is available on turf grass as transferable residue.
   * Oral postapplication exposure was estimated using the standard assumption for the initial fraction of residues available and assuming a 10% daily dissipation rate. 
   * The median surface area of both hands is 20 cm[2] for children.
   * It is assumed that there is a one-to-one relationship between the dislodgeable residues on the turf and on the surface area of the skin after contact.
   * The mean rate of hand-to-mouth activity is 20 times/hour for short-term exposure scenarios.
   * The saliva extraction factor is 50%.
   * The children (3-6 yrs) are assumed to weigh 15 kg.
   * Duration of exposure for children is assumed to be 2 hours per day for turf.

The following series of equations was used to estimate hand-to-mouth exposure; essentially, the turf transferable residue (TTR) is determined using default assumptions and the application rate, and the TTR is then used along with the frequency of mouthing, the time spent on turf, and the child body weight to determine the potential dose rate.  The NOAEL for the incidental oral endpoint is divided by the dose to determine the MOE.

TTRt = AR x F x (1-D)[t] x CF2 x CF3             

TTR	=	Turf Transferable Residue;
AR	= 	Application rate (lb ai/acre);
F	=	Fraction of ai available on turf (unitless) (0.05);
D	=	Fraction of residue that dissipates daily (unitless) (0.1);
t 	=	Postapplication day on which exposure is being assessed;
CF2	=	Weight unit conversion factor to convert the lbs ai in the application rate to ug for DFR
		value (4.54 x 10[8] ug/lb); and,
CF3	=	Area unit conversion factor to convert the surface area units (acre) in the application rate 
			to cm[2] for the DFR value (2.47 x 10[-8] acre/cm[2]).
  
PDR = TTR x SA x FQ x ET x SE x CF1	    
            BW
            		           
PDR	=	Potential dose rate (mg/day);
TTR	=	Turf transferable residue (ug/cm[2] turf);
SA	=	Surface area of the hands (20 cm[2]/event);
FQ	=	Frequency of hand-to-mouth activity (20 events/hr for short-term) ;
ET	=	Exposure time (hr/day);
CF1		=	Weight unit conversion factor to convert ug units in the TTR value to mg for the daily
			exposure (0.001 mg/ug for turf);
SE	=	Saliva Extraction Factor (50%); and,
BW	=	Body Weight (15 kg).

Hand-to-Mouth (HTM) Exposure and Risk Estimate

Short-term HTM exposure resulted in an MOE greater than the level of concern (MOEs >= 100) and therefore poses no risk of concern.  Table 6.2 provides a summary of the short-term HTM exposure estimate.

Table 6.2.  Amicarbazone Children's Short-term Non-Dietary Ingestion (Hand-to-Mouth) Exposure and Risk Estimate.
Product
TTR 1 (ug/cm[2])
SA (cm[2]/event)
FQ
SE
ET (hr/day)
CF1
BW (kg)
HTM Dose [2]
(mg/kg/day)
HTM MOE [3]
Amicarbazone DF Herbicide 
70% ai
(EPA Reg. No. 66330-46)
                                    0.2576
                                      20
                                      20
                                      0.5
                                       2
                                     0.001
                                      15
                                    0.00687
                                      910
   1. Turf Transferable Residues = (TTR) = 
   AR (0.46 lb ai/A) x F (0.05) x (1-D)[0] x CF2 (4.54E[8] ug/lb) x CF3 (2.47E[-8] acre /cm[2]) = 0.2576 ug/cm[2]
   2. HTM Dose = [TTRt x SA x FQ x ET x SE x CF1]/BW
   3. HTM MOE = NOAEL (6.28 mg/kg/day)/HTM Dose (mg/kg/day)

Non-Dietary Ingestion (Object to Mouth Ingestion and Soil Ingestion)

In addition to the HTM exposure assessed for children playing on treated lawns, HED also assessed potential exposure associated with mouthing objects that have come into contact with treated turf, as well as the potential for incidental ingestion of soil on treated turf.  These exposures were significantly lower than that estimated for HTM exposure.  HED used similar typical assumptions with respect to the amount of time spent on turf, the child body weight, and the application rate.  Standard assumptions were used to estimate the amount of pesticide retained on objects and in soil.  For example, HED has standard assumptions with respect to the amount of pesticide retained in the uppermost 1cm of soil, and for the fraction of residue that dissipates daily.

The estimated MOEs were 3,700 for object-to-mouth ingestion, and 270,000 for soil ingestion.  These risks associated with children playing on treated turf are not of concern.  Details regarding these calculations are available in the Z. Figueroa memo, D394924.

6.3	Combined Exposure

HED combines risk values resulting from separate exposure scenarios when it is likely they can occur simultaneously based on the use pattern and the behavior associated with the exposed population.  In evaluating combined residential uses of amicarbazone, HED reviewed all residential sources of exposure which consisted of: 1) adult inhalation handler (lawns only) exposure, and 2) child postapplication oral exposure.  

Since a dermal endpoint was not selected for amicarbazone, the only route of exposure for adults is through the inhalation route, and therefore, a combined residential exposure assessment is not applicable.  

The children's oral exposure estimate for the purpose of conducting an aggregate risk assessment is based on postapplication hand-to-mouth exposures only.  To include exposure from object-to-mouth and soil ingestion in addition to hand-to-mouth could result in a very conservative estimate of exposure since the assumptions for each of the individual oral exposure calculations are high end.

Table 6.3 identifies the residential scenarios and MOEs for adults and children for use in performing an aggregate exposure assessment as part of the amicarbazone human health risk assessment.  There are no risks of concern.

Table 6.3.  Amicarbazone Summary of Residential Exposure and Risk Estimates. 
Product
Use Site
Handler Inhalation MOE [1]
Postapplication Dermal MOE 
Hand-to-Mouth MOE [2]
Combined MOE
                                    Adult 
Amicarbazone DF Herbicide (DINAMIC 
70 WDG HERBICIDE)
                                 Lawns (turf)
                                     3,600
                                      NA
                                      NA
                                      NA
                                     Child
Amicarbazone DF Herbicide (DINAMIC 
70 WDG HERBICIDE)
                                 Lawns (turf)
                                      NA
                                      NA
                                      910
                                      NA
   1. See Table 6.1: Residential Handler Exposure and Risk (Low Pressure Handwand).
   2. See Table 6.2. Hand-To-Mouth Exposure and Risk.

6.4	Residential Bystander Postapplication Inhalation Exposure

Based on the Agency's current practices, a quantitative postapplication inhalation exposure assessment was not performed for amicarbazone at this time primarily because of the low acute inhalation toxicity (Toxicity Category IV), low vapor pressure (2.3 x 10[-8] mm Hg) and the proposed use rate (0.46 lb ai/A).  However, volatilization of pesticides may be a potential source of postapplication inhalation exposure to individuals nearby to pesticide applications.  The Agency sought expert advice and input on issues related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009, and received the SAP's final report on March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html).  The Agency is in the process of evaluating the SAP report and may, as appropriate, develop policies and procedures to identify the need for and, subsequently, the way to incorporate post-application inhalation exposure into the Agency's risk assessments.  If new policies or procedures are developed, the Agency may revisit the need for a quantitative postapplication inhalation exposure assessment for amicarbazone.

Although a quantitative residential postapplication inhalation exposure assessment was not performed, an inhalation exposure assessment was performed for residential handlers.  This exposure scenario is representative of a worse case inhalation exposure and should be considered protective of most postapplication inhalation exposure scenarios.

6.5	Spray Drift

Spray drift is always a potential source of exposure to residents near spraying operations.  This is particularly the case with aerial application, but, to a lesser extent, could also be a potential source of exposure from the ground application method employed for amicarbazone.  The Agency has been working with the Spray Drift Task Force, EPA Regional Offices and State Lead Agencies for pesticide regulation and other parties to develop the best spray drift management practices (see the Agency's Spray Drift website for more information at http://www.epa.gov/opp00001/factsheets/spraydrift.htm).  On a chemical by chemical basis, the Agency is now requiring interim mitigation measures for aerial applications that must be placed on product labels/labeling.  The Agency has completed its evaluation of the new database submitted by the Spray Drift Task Force, a membership of U.S. pesticide registrants, and is developing a policy on how to appropriately apply the data and the AgDRIFT computer model to its risk assessments for pesticides applied by air, orchard airblast and ground hydraulic methods.  After the policy is in place, the Agency may impose further refinements in spray drift management practices to reduce off-target drift with specific products with significant risks associated with drift.

The 0.46 lb ai/acre application rate for turf was modeled to estimate postapplication residential exposure of children.  As this rate is the same as the agricultural maximum single application rate (0.46 lb ai/A), this scenario is protective of any potential exposure of farm children via spray drift from agricultural applications of amicarbazone.

6.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and risks from three major sources: food, drinking water, and residential exposures.  In an aggregate assessment, exposures from relevant sources are added together and compared to quantitative estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated.  When aggregating exposures and risks from various sources, HED considers both the route and duration of exposure.  In the case of amicarbazone, acute and chronic aggregate risks result from exposure through food and water only.  For short-term risks, residential handlers' inhalation exposure and children's incidental oral exposures were combined with background exposure from food and water. 

7.1	Acute Aggregate Risk

Acute aggregate risk is equivalent to acute dietary exposure and risk, which is not of concern.  Refer to Section 5.4.3.

41.2 Short-Term Aggregate Risk

The residential handler exposure from applying amicarbazone using a low pressure handwand (Table 6.1) and children's postapplication oral exposure (Table 6.2) were combined with the chronic dietary exposure from the mostly highly exposed adult (General US population) and children's (all infants <1 year old) subpopulations (Table 5.4.5), respectively, to determine aggregate exposure and risk as shown in Table 7.2.  Despite the numerous conservative assumptions in developing these estimates, the MOEs are above the LOC of 100, and are not of concern.

Table 7.2.  Short-Term Aggregate Risk Calculations for Amicarbazone.
                                  Population
                                NOAEL mg/kg/day
                                    LOC[1]
                      Max Allowable Exposure[2] mg/kg/day
                   Average Food and Water Exposure mg/kg/day
                       Residential Exposure mg/kg/day[3]
                          Total Exposure mg/kg/day[4]
                Aggregate MOE (food, water, and residential)[5]
Adult (Handler)
                                     6.28
                                      100
                                    0.0628
                                   0.003889
                                    0.00175
                                   0.005639
                                     1,100
Child (Postapplication)
                                     6.28
                                      100
                                    0.0628
                                   0.010987
                                    0.00687
                                   0.0178587
                                      350
[1] The LOC is based on the standard inter- and intra- species uncertainty factors totaling 100.  The FQPA Safety Factor has been reduced to 1X.
[2] Maximum Allowable Exposure (mg/kg/day) = NOAEL/LOC
[3] Residential Exposure (Adult Handler) = Inhalation Exposure (Table 6.1).  Residential Exposure (Child Postapplication) = Hand-to-Mouth Exposure (Table 6.2).
[4] Total Exposure = (Avg. Food & Water Exposure + Residential Exposure)
[5] Aggregate MOE = [NOAEL/Total Exposure]

7.3	Chronic Aggregate Risk

Chronic aggregate risk is equivalent to chronic dietary exposure and risk, which is not of concern.  Refer to Section 5.4.4.

6.0 Cumulative Exposure/Risk Characterization

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

7.0 Occupational Exposure/Risk Characterization

9.1	Short-/Intermediate-Term Handler Risk

The proposed use pattern for the dry flowable (DF) product containing amicarbazone involves applying sprays after mixing with water or a liquid fertilizer.  No chemical-specific information was provided regarding the number of exposure days per year, but based on the proposed use pattern HED has assumed that short- and intermediate-term exposure durations can be expected for occupational handlers.

HED has assessed exposure for occupational handlers for the following scenarios:

   1) mixing/loading dry flowable for ground applications;
   2) applying sprays via open cab groundboom equipment; 
   3) mixing/loading/applying liquids with manually pressurized handwand (low-pressure) handwand;
   4) mixing/loading/applying liquids with a backpack sprayer; and,
   5) mixing/loading/applying liquids with mechanically pressurized handgun sprayer. 

No chemical-specific handler exposure data were submitted in support of the proposed use, and therefore HED relied on the best available surrogate data to complete the occupational handler assessment.  Sources of generic handler data, used as surrogate data in the absence of chemical-specific data, include the Pesticide Handlers Exposure Database Version 1.1 (PHED 1.1), the Agricultural Handler Exposure Task Force (AHETF) database, and the Outdoor Residential Exposure Task Force (ORETF) database.  Some of these data are proprietary (e.g., AHETF data, MRID No. 44339801), and subject to the data protection provisions of FIFRA.  The standard values recommended for use in predicting 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/science/handler-exposure-table.pdf), which, along with additional information on HED policy on use of surrogate data, including descriptions of the various sources, can be found at http://www.epa.gov/pesticides/science/handler-exposure-data.html.

The proposed product label involved in this assessment directs applicators and other handlers to wear long sleeved shirt and long pants, shoes plus socks, and chemical-resistant gloves.  HED typically assesses handler exposure using "baseline" clothing assumptions, and if risks of concern are identified, the use of personal protective equipment (PPE) may be incorporated into the exposure assessment.  In the case of amicarbazone, there is no toxicity via the dermal route, and only inhalation exposures were assessed.  No additional PPE (i.e., respirators) were needed to achieve MOEs above the LOC of 100.

Standard assumptions were used with respect to body weight for adult handlers (70 kg), the exposure duration (i.e., short- and intermediate-term), and the area treated for various types of application equipment and application sites.  In conjunction with these standard values, HED used the maximum application rates from the proposed label directions.  Each of the risks is presented as a MOE, or the ratio of the NOAEL to the calculated daily dose.

Table 9.1 shows the results of HED's exposure and risk assessment for occupational handlers.  Based on a personal communication with the registrant, the proposed label will prohibit the use of a mechanically pressurized handgun for application to Christmas trees.  The MOEs shown in the table are all significantly higher than HED's LOC of an MOE of 100, with the lowest MOE of 1300 identified for handlers conducting mixing/loading activities for application to sod farms.  Risk associated with this and all other scenarios is not of concern.

Table 9.1.  Amicarbazone.  Short- and Intermediate-Term Exposure and Risk for Occupational Handlers.
                               Exposure Scenario
                                Crop or Target
                         Inhalation Unit Exposure [1]
                                    Maximum
                             Application Rate [2]
                       Acres or Amount Treated Daily [3]
                                  Inhalation
                                       
                                       
                               Mitigation Level
                                       
                                       
                                   Dose [4]
                                    MOE [5]
                                 Mixer/Loader
(1)Mixer/Loader - Ground  -  
Dry Flowable
Golf courses
                              0.00896 (Baseline)
                                    (AHETF)
                                     0.46
                                    lb ai/A
                                     40 A
                                    0.00235
                                     2,700

Sod farms (turf), Christmas trees
                                       
                                       
                                       
                                       
                                       

                                       
                                       
                                     80 A
                                    0.00471
                                     1,300
                                  Applicator
(2)Applicator - Open Cab Groundboom
Golf courses 
                              0.00034 (Baseline)
                                    (AHETF)
                                     0.46
                                    lb ai/A
                                     40 A
                                   0.0000894
                                    70,000

Sod farms (turf), Christmas trees
                                       
                                       
                                     80 A
                                   0.000178
                                    35,000
                            Mixer/Loader/Applicator
(3)Backpack Sprayer
Christmas trees
(Ground-Directed)
                              0.00258 (Baseline)
                                (MRID 44339801)
                                     0.023
                                   lb ai/gal
                                    5 gals
                                   0.0000042
                                   1,500,000

Turf, golf course, sod farms, parks, recreation and athletic fields 
                                       
                                       
                                       
                                       
                                       
(4)Manually pressurized Handwand
Turf, Christmas trees
                                0.03 (Baseline)
                                    (PHED)
                                     0.023
                                   lb ai/gal
                                    5 gals
                                   0.000049
                                    130,000
               (5)Mechanically-pressurized Handgun Sprayer (WDG)
                              Turf, golf courses
                                  (Broadcast)
                               0.042 (Baseline)
                                    (ORETF)
                                     0.46
                                    lb ai/A
                                      5 A
                                    0.00138
                                     4,500
   [1] Occupational Pesticide Handler Unit Exposure Surrogate Reference Table (September 26, 2011).
   2 Based on proposed label (Reg. No. 66330-46).
   3 Exposure Science Advisory Council Policy #9.1.
   4 Inhalation Dose = [Inhalation Unit Exposure (mg/kg) x Application Rate (lb ai/acre or gal) x Acres or Amount Treated per day (A/day or gal/day)] /BW (70 kg)
   [5] Inhalation MOE = Inhalation NOAEL (6.28 mg/kg/day)/ Inhalation Dose (mg/kg/day)

9.2	Short-Term Postapplication Risk

9.2.1	Dermal Postapplication Risk

 Dermal postapplication exposure assessments were not performed because a dermal endpoint was not selected for amicarbazone.
 
Restricted Entry Interval
The restricted entry interval (REI) listed on proposed label is based on the acute toxicity of the technical material.  Amicarbazone exhibits low acute toxicity and is classified as Toxicity Category III for eye irritation and Category IV for acute dermal and dermal irritation.  It is not a dermal sensitizer.  Acute toxicity Category III and IV chemicals require a 12-hour REI.  Therefore, the 12-hour REI which appears on the proposed labels is considered appropriate. 

9.2.2	Inhalation Postapplication Risk

Based on the Agency's current practices, a quantitative postapplication inhalation exposure assessment was not performed for amicarbazone at this time primarily because it has a low vapor pressure (2.3 x 10[-8] mm Hg), it is applied at an application rate of 0.46 lbs ai/A, and it is not projected to be applied via typically high inhalation exposure application equipment (e.g., airblast and aerial equipment).  However, volatilization of pesticides may be a potential source of postapplication inhalation exposure to individuals nearby to pesticide applications.  The Agency sought expert advice and input on issues related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009.  The Agency received the SAP's final report on March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html).  The Agency is in the process of evaluating the SAP report and may, as appropriate, develop policies and procedures to identify the need for and, subsequently, the way to incorporate postapplication inhalation exposure into the Agency's risk assessments.  If new policies or procedures are put into place, the Agency may revisit the need for a quantitative postapplication inhalation exposure assessment for amicarbazone.

Although a quantitative occupational postapplication inhalation exposure assessment was not performed, an inhalation exposure assessment was performed for occupational/commercial handlers.  Handler exposure resulting from application of pesticides outdoors is likely to result in higher exposure than postapplication exposure.  Therefore, it is expected that these handler inhalation exposure estimates would be protective of occupational postapplication inhalation exposure scenarios.

10.0	References

D288216.  Amicarbazone: HED Human Health Risk Assessment for New Food Use Herbicide on Field Corn.  K. Kosick, 8/10/2005.

D384659.  Estimated Drinking Water Concentrations of Parent Amicarbazone and its Degradates for New Use on Turf and Conifers.  A. Shelby, 9/7/11.

D394925.  Amicarbazone Acute and Chronic Aggregate Dietary (Food and Drinking Water) Exposure and Risk Assessments for the Section 3 Registration Action on Turfgrass.  D. Dotson, 10/7/2011.

D394952.  Amicarbazone. Storage Stability of Amicarbazone and its Residues of Concern in Field Corn Forage, Grain, and Stover. Evaluation of Submitted Study.  D. Dotson, 11/1/11

D388103.  Amicarbazone: Review of a 28-Day Dietary Immunotoxicity Study in Rats. V. Chen, 11/30/11.

D394924.  Amicarbazone.  Occupational and Residential Exposure Assessment for Proposed Uses on Turf, Christmas Tree Farms, Non-Crop Areas and on Conifers in Nurseries.  Z. Figueroa, 11/30/11.
Appendix A.  Toxicology Profile and Executive Summaries

A.1	Toxicology Data Requirements
The requirements (40 CFR 158.340) for food use for amicarbazone are in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

                                     Study
                            Technical Amicarbazone

                                   Required
                                   Satisfied
870.1100    Acute Oral Toxicity	
870.1200    Acute Dermal Toxicity	
870.1300    Acute Inhalation Toxicity	
870.2400    Primary Eye Irritation	
870.2500    Primary Dermal Irritation	
870.2600    Dermal Sensitization	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.3100    Oral Subchronic (rodent)	
870.3150    Oral Subchronic (nonrodent)	
870.3200    21-Day Dermal	
870.3250    90-Day Dermal	
870.3465    28-Day Inhalation	
                                      yes
                                      yes
                                      yes
                                      no
                                      no
                                      yes
                                      yes
                                      yes
                                      --
                                      no
870.3700a  Developmental Toxicity (rodent)	
870.3700b  Developmental Toxicity (nonrodent)	
870.3800    Reproduction	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.4100a  Chronic Toxicity (rodent)	
870.4100b  Chronic Toxicity (nonrodent)	
870.4200a  Oncogenicity (rat)	
870.4200b  Oncogenicity (mouse)	
870.4300    Chronic/Oncogenicity	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.5100    Mutagenicity -- Gene Mutation - bacterial	
870.5300    Mutagenicity -- Gene Mutation - mammalian	
870.5xxx    Mutagenicity -- Structural Chromosomal Aberrations	
870.5xxx    Mutagenicity -- Other Genotoxic Effects	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.6100a  Acute Delayed Neurotoxicity (hen)	
870.6100b  90-Day Neurotoxicity (hen)	
870.6200a  Acute Neurotoxicity Screening Battery (rat)	
870.6200b  90-Day Neurotoxicity Screening Battery (rat)	
870.6300    Develop. Neurotoxicity	
                                      no
                        no                         yes
                                      yes
                                      yes
                                      --
                       --                           yes
                                      yes
                                      yes
870.7485    General Metabolism	
870.7600    Dermal Penetration	
870.7800    Immunotoxicity	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
Special Studies for Ocular Effects
         Acute Oral (rat)	
         Subchronic Oral (rat)	
         Six-month Oral (dog)	
                                       
                                      no
                                      no
                                      no
                                       
                                      --
                                      --
                                      --

A.2	Toxicity Profiles

Table A.2.1	Acute Toxicity Profile  -  Amicarbazone Technical
Guideline No.
Study Type
MRID(s)
                                    Results
                               Toxicity Category
870.1100
Acute oral [rat]
                                   45121504
Males LD50 > 2050 mg/kg ;
Females LD50 = 1015 mg/kg
                                      III
870.1200
Acute dermal [rat]
                                   45121503
LD50 > 5000 mg/kg
                                      IV
870.1300
Acute inhalation [rat]
                                   45121506
LC50 = 2.030 mg/L
                                      IV
870.2400
Acute eye irritation [rabbit]
                                   45121510
Eye irritation including corneal opacity at 24 hr, cleared by Day 7
                                      III
870.2500
Acute dermal irritation [rabbit]
                                   45121509
No primary irritation observed
                                      IV
870.2600
Skin sensitization [guinea pig]
                                   45121505
                                   45121628
Not a dermal sensitizer
                                      N/A

Table A.2.2	Subchronic, Chronic and Other Toxicity Profile
                                Guideline No. 
                                  Study Type
                    MRID No. (year)/ Classification /Doses
                                    Results
870.3100

90-Day oral toxicity (Fischer -344 rat)
45121523 (1997)
Acceptable/Guideline
0, 100, 250, 500, 1000, 2500, 5000ppm

M: 0, 6.9, 18, 33, 67, 182, 354 mg/kg/day

F: 0, 7.9, 21, 38, 78, 201, 397mg/kg/day
NOAEL = 33/38 mg/kg/day [m/f]
LOAEL = 67/78 mg/kg/day [m/f] based on decreased body weight in females and cumulative body weight gain, decreased red cell indices, clinical chemistry alterations (increased cholesterol, T4, T3, N-demethylase in males and O-demethylase in females), increased relative liver weights in males and histopathology of the liver (minimal hepatocytomegaly) and spleen (minimal pigmentation) in males.
870.3150

90-Day oral toxicity (beagle dog)
45121524 (1998)
Acceptable/guideline

0, 200, 800, or 2000 ppm

M: 0, 6.74, 27.03, or 57.40 mg/kg/day

F: 0, 6.28, 24.99 (25), or 62.11mg/kg/day
NOAEL = 6.28 mg/kg/day
LOAEL = 24.99 mg/kg/day (25 mg/kg/day) based on increased thyroid vacuolization and decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights, and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase, and O-demethylase in both sexes.
870.3200

21/28-Day dermal toxicity (Sprague-Dawley rat)
45121525 (1998)
Acceptable/Guideline 

0, 200, 500, 1000 mg/kg/day
NOAEL = 1000 mg/kg/day
LOAEL = Not Observed
870.3700a

Prenatal developmental in (Sprague-Dawley rat)
45121530 (1999)
Acceptable/Guideline

0, 15, 100, 300 mg/kg/day
Maternal NOAEL = 15 mg/kg/day
LOAEL = 100 mg/kg/day based on decreased body weight/body weight gain and food consumption, and increased incidences of hard stools.
Developmental NOAEL = 15 mg/kg/day
LOAEL = 100 mg/kg/day based on multiple skeletal development retardations (incomplete ossification/unossification was observed in parietal bones, interparietal bones, supraoccipital bones, squamosal bones, zygoma, pubis, xiphoid, and fontanelle
870.3700b

Prenatal developmental in (Himalayan rabbit)
45121627 (1999)
Acceptable/Guideline

0, 5, 20, 70 mg/kg/day

Maternal NOAEL = 5 mg/kg/day
LOAEL = 20 mg/kg/day based on decreased body weight gain during treatment.
Developmental NOAEL = 20 mg/kg/day
LOAEL = 70 mg/kg/day based on decreased fetal body weight, and increased incidences of incomplete ossification of the 5[th] medial phalanx (bilateral) and the 13[th] caudal vertebra, and slightly thick ribs.
870.3800

Reproduction and fertility effects
(Sprague-Dawley rat)
45121625 (1998)
Acceptable/Guideline

0, 100, 500, 1000 ppm

M: 0, 6.4, 33.9, 73.2 mg/kg/day

F: 0, 7.3, 38.7, 84.0 mg/kg/day
Parental/Systemic NOAEL = 6.4/7.3 mg/kg/day
LOAEL = 33.9/38.7 mg/kg/day based on decreased body weight/body weight gain in both sexes.
Reproductive NOAEL = 73.2/84.0 mg/kg/day
LOAEL = Not Observed 
Offspring NOAEL = 6.4/7.3 mg/kg/day
LOAEL = 33.9/38.7 mg/kg/day based on decreased pup body weight and overall decreased body weight gain.
870.4100a

Chronic toxicity
(Fischer-344 rat)
45121512 (1999)
Acceptable/Guideline

0, 50, 500, 1250/1000 ppm

M: 0, 2.3, 25.3, 67 mg/kg/day

F: 0, 2.7, 29.5, 65 mg/kg/day
NOAEL = 2.3/2.7 mg/kg/day
LOAEL = 25.3/29.5 mg/kg/day based on decreased body weight in females and body weight gain in both sexes.

No evidence of carcinogenicity

At the doses tested there was not a treatment related increase in tumor incidence when compared to control.  Dosing was considered adequate based on decreased body weight in females and body weight gain in both sexes.
870.4100b

Chronic toxicity (beagle dog)
45121529 (1999)
Acceptable/Guideline

0, 75, 100, 300, 1200 ppm

M: 0, 1.6, 2.5, 8.9, 31.5 mg/kg/day

F: 0, 1.8, 2.3, 8.7 34.6mg/kg/day
NOAEL =2.5/2.3 mg/kg/day
LOAEL = 8.9/8.7 mg/kg/day based on effects on the liver, including increased absolute and relative liver weights, and O-demethylase in males ; increased globulin and cytochrome P-450 in females; and increased triglycerides and cholesterol in both sexes.

870.4300a

Combined chronic toxicity/carcinogenicity
(Fischer-344 rat)
45121512 (1999)
Acceptable/Guideline

0, 50, 500, 1250/1000 ppm

M: 0, 2.3, 25.3, 67 mg/kg/day

F: 0, 2.7, 29.5, 65 mg/kg/day
NOAEL = 2.3/2.7 mg/kg/day
LOAEL = 25.3/29.5 mg/kg/day based on decreased body weight in females and body weight gain in both sexes.

No evidence of carcinogenicity

At the doses tested there was not a treatment related increase in tumor incidence when compared to control.  Dosing was considered adequate based on decreased body weight in females and body weight gain in both sexes.
870.4300b

Carcinogenicity
(CD-1 mouse)
45121604 (1999)
Acceptable/Guideline

0, 100, 1500, 4000 ppm

M: 0, 15.7, 244.7, 709.0 mg/kg/day

F: 0, 17.9, 275.0, 806.3 mg/kg/day
NOAEL = 244.7/275.0 mg/kg/day
LOAEL = 709.0/806.3 mg/kg/day based on decreased body weight and body weight gain in both sexes, and subclinical anemia, and hemosiderin pigmentation of the spleen in males.

no evidence of carcinogenicity

At the doses tested there was not a treatment related increase in tumor incidence when compared to control.  Dosing was considered adequate based on decreased body weight and body weight gain in both sexes, and subclinical anemia, and hemosiderin pigmentation of the spleen in males.
Gene Mutation
870.5100 
Bacterial reverse gene mutation (S. typhimurium)
45121617 (1999)
Acceptable/Guideline

0, 16, 50, 158, 500, 1581, or 5000 g/plate in the presence and absence of S9-activation
There was no evidence of induced mutant colonies over background in strains TA98, TA100, TA102, TA1535, TA1537.
Gene Mutation
870.5100 
Bacterial reverse gene mutation (S. typhimurium)
45121616 (1996)
Acceptable/Guideline

0, 16, 50, 158, 500, 1581, or 5000 g/plate in the presence and absence of S9-activation
There was no evidence of induced mutant colonies over background in strains TA98, TA100, TA102, TA1535, TA1537.
Gene Mutation
870.5100 
Bacterial reverse gene mutation (S. typhimurium)
45121519 (1995)
Acceptable/Guideline

0, 16, 50, 158, 500, 1581, or 5000 g/plate in the presence and absence of S9-activation 
There was no evidence of induced mutant colonies over background in strains TA98, TA100, TA102, TA1535, TA1537.
Gene Mutation
870.5300 
Mammalian forward gene mutation (V79 Chinese hamster lung fibroblast cells)
45121514 (1997)
Acceptable/Guideline

0, 250, 500, 1000, 2000, or 4000 g/mL in the presence and absence of S9 activation for 5 hours 

4000 g/mL was the limit of solubility
There was no evidence that MKH3586 induced mutant colonies over background in the presence or absence of S9-activation.
Cytogenetics 
870. 5375
In vitro mammalian chromosome aberration test (V79 Chinese hamster lung fibroblast cells)
45121513 (1997)
Acceptable/Guideline

0, 1000. 2000, 4000, or 5000 g/mL for 4 hours in the presence and absence of S9-activation
There was no evidence of chromosome aberration induced over background in the presence or absence of S9-activation.
Other Effects 
870.5395 
Mammalian  in vivo erythrocyte micronucleus test (Hsd/Win NMRI mouse)
45121515 (1997)
Acceptable/Guideline

100 mg/kg

harvest times 16, 24, and 48 hours post dosing
There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow at any treatment time.
870.6200a

Acute neurotoxicity screening battery (Fischer-344 rat)
45121528 (1996)
Acceptable/Guideline in conjunction with 45121527

M: 0, 20, 150, 600 mg/kg/day

F: 0, 20, 100, 400 mg/kg/day
NOAEL = 10 mg/kg/day
LOAEL = 20 mg/kg/day based on eyelid ptosis, decreased approach response (both sexes), and red nasal staining in males.

A series of acute neurotoxicity studies were performed, the NOAEL for this study comes from 45121527.

870.6200b

Subchronic neurotoxicity screening battery (Fischer-344 rat)
45121532 (1999)
Acceptable/Guideline

0, 100, 500, 1000 ppm

M: 0, 6.7, 33.4, 66.5 mg/kg/day

F: 0, 7.8, 38.2, 75.8 mg/kg/day
Female 
NOAEL = 7.8 mg/kg/day
LOAEL = 38.2 mg/kg/day based on decreased body weight and overall body weight gain in females. 

Male
NOAEL = 66.5 mg/kg/day
 LOAEL = Not observed for males.

870.6300

Developmental neurotoxicity (Wistar rat)
45441301 (2001)

0, 100, 500, 1000 ppm

(gest): 0, 8, 39, 91 mg/kg/day

(lact): 0, 17, 84, 177 mg/kg/day
Maternal NOAEL = 8 mg/kg/day
LOAEL = 39 mg/kg/day based primarily on decreased feed efficiency (combination of decreased body weight gain and increased food consumption) during lactation.
Offspring NOAEL = 39 mg/kg/day
LOAEL = 91 mg/kg/day based on decreased body weight gain.
870.7485

Metabolism and pharmacokinetics
(Fischer-344 rat)
45121701 (1997)
Acceptable/guideline

MKH 3586 (amicarbazone)
95% of the radioactive dose was recovered within 72 hours following dosing.  The majority of the dose was recovered from the urine within 24 hours (64%), indicating substantial absorption.  Fecal excretion accounted for 27% of the dose within 24 hours.  Major metabolites were DA MKH, N-methyl DA MKH, and decarboxamide.
870.7485
Metabolism and pharmacokinetics
(Fischer-344 rat)
45121634 (1999)
Acceptable/Guideline

4-methyl MKH 3586 (soil metabolite)
91% of the radioactive dose was recovered within 96 hours.  Urinary excretion accounted for 70% of the radioactive dose within 12 hours, showing substantial absorption.  Only 8% of the radioactive dose was excreted via the feces within 24 hours.
870.7800
Immunotoxicity
(Female Fischer-344 rat)
48407504 (2011)
Acceptable/guideline
0, 250, 1000, 2500 ppm
0, 20, 81, 195 mg/kg/day

Immunotoxicity NOAEL = 81 mg/kg/day
Immunotoxicity LOAEL = 195 mg/kg/day, based on decreased spleen weight, total spleen cells/spleen and suppressed SRBC response.
Tentative systemic NOAEL = 81 mg/kg/day 
Tentative systemic LOAEL = 195 mg/kg/day, based on decreased body weight and body weight gain, food consumption and absolute spleen weight.
Special studies
Subchronic mechanistic feeding study (Fischer-344 rat)
45121603 (1999)
Acceptable/Nonguideline

0, 50, 1250, or 2500 ppm for 10 weeks

M:0, 0.8, 19.4, 40.0 mg/kg/day

F: 0, 0.6, 19.4, 28.8 mg/kg/day
Thyroid hormones were increased in the >=19.4 mg/kg/day females and 40.0 mg/kg/day males.  However, thyroid to blood ratios of [125]I in treated groups were comparable to negative controls, indicating there was no impairment of thyroid hormone synthesis.  Thus, the differences in thyroid hormones are probably due to metabolism at an extra-thyroidal site.  The liver was implicated as this site because liver weights and UDP-glucuronosyltransferase activity were increased.
Special studies
In vitro studies on enzymes of thyroid hormone regulation 
45121618 (1998)
Acceptable/Nonguideline
MKH 3586 does not affect the iodide organification step of thyroid hormone synthesis or the peripheral metabolism of thyroid hormones via Type I or Type II deiodinases in vivo.  These findings support the subchronic mechanistic studies in rats which indicate that up-regulation of UDP-glucuronosyl transferase in the liver may account for alterations in thyroid hormone profile.
Special studies
Nonguideline behavioral study  (HsdCbp WU rat)
45121511, 45121521, 45121629 (1997)

Acceptable/Nonguideline

0, 1.0, 2.5, 5, 10, 20, or 100 mg/kg
The following clinical signs were observed: sedation, ptosis, salivation (single animals affected except 2 for salivation) at 10 mg/kg/day and above.  Additionally at the HDT, piloerection, Straub phenomenon, and prone position were observed.  The effects were observed at 30 minutes post dose, and no effect was observed at 150 minutes post dose, with the higher dose groups showing greater persistence of effects.  A dose- and time-dependent effect was demonstrated on motor activity - decreased travel distance, increased resting time, and decreased rearing.  
Special studies
Nonguideline study of central nervous system safety pharmacology  (HsdWin: NMRI mouse)
45121522 (1997)
Acceptable/Nonguideline

0, 1, 20, 100 mg/kg/day
The data indicate that a single dose of MKH 3586 at 100 mg/kg causes minimal CNS functional impairment, characterized by increased reaction times to nociceptive stimuli, reduced traction force, impaired motor coordination, sedation, partial ptosis, and a mild anticonvulsive effect.

A.3	Hazard Identification and Endpoint Selection

A.3.1	Acute Reference Dose (aRfD)  -  General Population Including Females age 13-49 and Infants and Children

Study Selected:  Acute Oral Neurotoxicity  -  Rat 
MRID No.:   45121527, -28
Executive Summary:  See Appendix A, Guideline [§ 870.6200a] 
Dose and Endpoint for Risk Assessment: NOAEL = 10 mg/kg/day (MRID 45121527) based on eyelid ptosis and decreased approach response (both sexes) and red nasal staining (males) seen at LOAEL = 20 mg/kg/day (MRID 45121528).
Comments about Study/Endpoint/Uncertainty Factors:  This study was selected because dosing was oral and effects were observed following a single dose.  No other studies (including the developmental toxicity studies) showed effects that could be considered the result of a single dose.  The study is protective of effects in all populations.  A combined UF of 100x (inter- and intra-species uncertainty) was utilized.

A.3.2	Chronic Reference Dose (cRfD)

Study Selected:  Chronic Dietary - Rat and Chronic Dietary  -  Dog (co-critical studies)
MRID Nos.:   45121512 (rat); 45121529 (dog)
Executive Summary:  See Appendix A, Guideline [§ 870.4100a and b] 
Dose and Endpoint for Risk Assessment: NOAEL = 2.3 mg/kg/day based on decreased body weight and weight gain in rats at LOAEL = 25.3 mg/kg/day and liver effects in dogs at LOAEL = 8.7 mg/kg/day.
Comments about Study/Endpoint/Uncertainty Factors:   These studies were selected because they both had the same NOAEL, were of the appropriate route (oral) and duration (chronic) and provided the most sensitive point of departure, including being protective for developmental, reproductive and neurotoxic effects.  The selected NOAEL is therefore protective of all populations.  A combined UF of 100x (inter- and intra-species uncertainty) was utilized.

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

Study Selected:  Subchronic oral toxicity  -  Dog 
MRID No.:   45121524
Executive Summary:  See Appendix A, Guideline [§ 870.3150] 
Dose and Endpoint for Risk Assessment: NOAEL = 6.28 mg/kg/day based on increased thyroid vacuolization and decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights, and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase and O-demethylase in both sexes seen at LOAEL = 24.99 mg/kg/day (25 mg/kg/day).
Comments about Study/Endpoint/Uncertainty Factors:  The route of exposure (oral) and study duration (90-day) are appropriate for this risk assessment.  This study was selected because it identified numerous effects and provided a sensitive point of departure that was consistent with the findings in other studies of similar duration that were under consideration in for this exposure scenario:  offspring NOAEL in the rat reproductive toxicity study (6.4 mg/kg/day, with a LOAEL of 33.9 mg/kg/day) and maternal NOAEL in the rabbit developmental toxicity study (5 mg/kg/day, with a LOAEL of 20 mg/kg/day), both based on decreased body weight/weight gain, and NOAEL of 7.8 mg/kg/day in the subchronic rat neurotoxicity study, based on decreased body weight and gain in females at 38.2 mg/kg/day.  The slightly higher NOAEL of the dog subchronic study compared to the reproductive and developmental studies was likely due to dose spacing.  All other NOAELs identified in the database for studies of the appropriate duration were higher.  A combined UF of 100x (inter- and intra-species uncertainty) was utilized.

A.3.4	Dermal Exposure (All Durations) 

An endpoint for short- and intermediate-term dermal exposure was not selected.  No systemic or local dermal toxicity was observed at the high dose of 1000 mg/kg/day (limit dose) in the rat 21-day dermal toxicity study.   A comparison of the LOAELs from several other rat studies supported the conclusion that amicarbazone was poorly absorbed (see Section 4.2.1).

A.3.5	Inhalation Exposure (Short-, Intermediate- and Long-Term)
 
Study Selected:  Subchronic Oral Toxicity  -  Dog 
MRID No.:   45121524
Executive Summary:  See Appendix A, Guideline [§ 870.3150] 
Dose and Endpoint for Risk Assessment: NOAEL = 6.28 mg/kg/day based on increased thyroid vacuolization, decreased food consumption and glucose (females); increased platelets, phosphate, bile acids, absolute/relative liver weights and lymphoid hyperplasia of the gall bladder (males); and decreased albumin and increased triglycerides, N-demethylase and O-demethylase in both sexes seen at LOAEL = 24.99 mg/kg/day (25 mg/kg/day).
Comments about Study/Endpoint/Uncertainty Factors:  Subchronic inhalation data were not available for amicarbazone; therefore, an oral study of the appropriate duration was selected.  The endpoint selected for this exposure assessment is of the appropriate duration and is considered protective for potential effect to all populations, including infants and children and females of reproductive age (see Comments in A.3.5 for Incidental Oral Exposure).  In the absence of inhalation data, inhalation toxicity was assumed to be equivalent to oral toxicity.  A combined UF of 100x (10x each for inter- and intra-species uncertainty) was utilized.

A.4	Executive Summaries

A.4.1	Subchronic Toxicity

	870.3100	90-Day Oral Toxicity  -  Rat

      In a subchronic oral toxicity study (MRID 45121523), MKH 3586 (amicarbazone; 98.2% a.i., Lot/Batch #: 17004/93) was administered to 15 CDF[F-344]/BR rats/sex/group in the diet at dose levels of 0, 100, 250, 500, 1000, 2500, or 5000 ppm (equivalent to 0/0, 6.9/7.9, 18/21, 33/38, 67/78, 182/201, and 354/397 mg/kg/day [M/F], respectively) for 13 weeks.  An additional 15 rats/sex/group were similarly treated at 0 and 5000 ppm and further observed during a 4-week recovery period following dosing.

      No adverse treatment-related effects were noted in mortality, clinical signs, ophthalmoscopy, or gross pathology.

      Body weight was decreased (p<=0.05) during Weeks 2-13 in the >=1000 ppm females (decr 4-21%) and in the >=2500 ppm males (decr 6-20%).  Overall (Weeks 1-13) body weight gain (calculated by reviewers) was decreased at >=1000 ppm in both sexes (decr 17-62%).  Food consumption was decreased (p<=0.05) throughout the study at >=2500 ppm in both sexes (decr 6-24%).

      In the liver, increases (p<=0.05, unless otherwise noted) were observed in absolute weight in the >=2500 ppm females (incr 13-15%, not statistically significant) and in the 5000 ppm males (incr 18%) and in relative (to body) weight in the >=1000 ppm females (incr 11-41%) and in the >=2500 ppm males (incr 21-43%).  Cholesterol was increased at >=1000 ppm in both sexes (incr 27-298%).  In the tissue samples collected at termination, O-demethylase activity was increased in the >=1000 ppm females (incr 33-89%) and in the >=2500 ppm males (incr 30 [not statistically significant]-60%), and N-demethylase activity was decreased in the >=1000 ppm males (decr 31-37%).  Minimal to marked hepatocytomegaly was observed in the >=1000 ppm males (9-10 treated vs 0 controls) and in the >=2500 ppm females (8-10 treated vs 0 controls)

      In the thyroid, increased incidence (# affected vs 0 controls) of minimal follicular cell hyperplasia was observed at >=2500 ppm in the males (2-7) and females (4-7).  Increased T4 activity was observed in the >=1000 ppm males, and increased T3 activity was observed in the >=1000 ppm males and in the >=2500 ppm females.

      In the spleen, increased incidence of hemosiderin pigmentation was observed in the >=1000 ppm males (4-5 treated vs 1 control) and in the >=2500 ppm females (3-10 treated vs 0 controls).  The following differences (p<=0.05, unless otherwise noted) in hematology parameters were observed: (i) red blood cells, hemoglobin, and hematocrit were decreased in the >=1000 ppm males (decr 3-11%); (ii) hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were decreased in the >=1000 ppm females (decr 3-15%); (iii) reticulocytes were increased at >=2500 ppm in the males (incr 33 [not statistically significant]-144%) and in the females (incr 47 [not statistically significant]-200%); (iv) platelets were increased in the >=2500 ppm males (incr 14-32%); and (v) MCH and MCV were decreased in the >=2500 ppm males (decr 2-4%).  Increased urinary pH was noted in the >=1000 ppm females, and increased urinary leukocytes were observed in the >=2500 ppm males and females.

      In the >=2500 ppm females, increased incidence (# affected vs 0 controls) of minimal to moderate corticomedullary atrophy of the bone (3-10), and minimal to moderate atrophy of the bone marrow (3-8) were observed.

      Additionally at 5000 ppm, red blood cells were decreased in the females, total protein was increased in the males and females, and globulins were increased in the males.   The following histopathological effects were observed:  minimal diffuse cytoplasmic vacuolization of the cortical epithelium cells of the adrenal glands in the males (10) and minimal cytoplasmic vacuolization of the pancreas in the males (10) and females (5).

      The only finding below 1000 ppm was increased (p<=0.05) cholesterol at 250 (incr 20-31% in males) and 500 ppm (incr 23-47% in both sexes).  In the 5000 ppm recovery group, all treatment-related effects showed signs of recovery; however, most parameters were still different (p<=0.05) from controls.

      The LOAEL is 1000 ppm (equivalent to 67/78 mg/kg/day [M/F]) based on decreased body weight (females) and overall (Weeks 1-13) body weight gains, decreased red cell indices, clinical chemistry (increased cholesterol, T4 [males], T3 [males], o-demethylase [females], and n-demethylase [males]), increased relative (to body) liver weight in females, and histopathological effects in the males (minimal hepatocytomegaly and minimal pigmentation in the spleen).  The NOAEL is 500 ppm (equivalent to 33/38 mg/kg/day [M/F]).

      This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3100a; OECD 408) for a subchronic oral toxicity study in the rat.

	870.3100	90-Day Oral Toxicity - Mouse

      Not submitted.

	870.3150	90-Day Oral Toxicity  -  Dog

      In a subchronic oral study (MRID 45121524), MKH 3586 (Amicarbazone technical; 98.2-99.1% a.i., Lot/batch #: 05362/0005) was administered to 4 beagle dogs/sex/dose in the diet at doses of 0, 200, 800, or 2000 ppm (equivalent to 0/0, 6.74/6.28, 27.03/24.99, 57.40/62.11 mg/kg/day [M/F]) for up to 90 days.  There were no treatment-related effects on survival, neurological evaluations, urinalysis, or ophthalmoscopy. 

      Food consumption was decreased by 22-43% (p<=0.05):  in the 800 ppm females on Days 6, 12, and 28; in the 2000 ppm males on Day 15; and in the 2000 ppm females on Days 2, 6, 12, and 28.  Additionally at 2000 ppm, one male was described as thin beginning on Day 21.  Body weights were decreased by 6-15% (not significant [NS]) in females throughout treatment, and overall (Days 0-91) body weight gains (calculated by reviewers) were decreased by 29-40% in both sexes at this dose.  Electrocardiography at the pre-terminal examination revealed the P-R interval was increased by 29% (p<=0.05) in the 2000 ppm males.  In the males, platelets were increased (p<=0.05) at >=800 ppm; and, additionally at 2000 ppm, activated partial thromboplastin time and hemoglobin were decreased (p<=0.05).

      Treatment-related effects were observed in the liver and gall bladder.  In the liver tissue samples, N-demethylase and O-demethylase activities were increased (p<=0.05) over controls in both males of >= 800 ppm and females of  >=200 ppm. N-demethylase and O-demethylase activities were increased (p<=0.05) by 62 and 42% over the controls, respectively,  in females of the 200 ppm group.  N-demethylase and O-demethylase activities were increased (p<=0.05) over controls by 212 and 79%, respectively, in males and by 195 and 142 %, respectively, in females, of the 800 ppm group.  At 2000 ppm, the increase (p<=0.05) in  N-demethylase and O-demethylase activities was 260 and 129%, respectively, in males, and 239 and 83%, respectively, in females.  Although terminal body weights were decreased by 13% (NS) in the 2000 ppm females, increases of 24-58% were noted in absolute (NS) and relative (p<=0.05) liver weights in the >=800 ppm males and 2000 ppm females.  Additionally at 2000 ppm, increased incidence (NS, unless otherwise noted) of the following microscopic findings were observed in the liver vs 0/4 controls: (i) bile pigmentation in males (1/4); (ii) individual cell necrosis in males (3/4) and females (4/4; p<=0.05); and hypertrophy in females (1/4).  Lymphoid hyperplasia of the gall bladder was increased (NS) in incidence and severity in the >=200 ppm males (2/4 to 3/4 treated vs 1/4 controls).  Additionally at 2000 ppm, abnormal texture of the gall bladder was noted (vs 0 controls) in males (1/4 treated) and females (4/4 treated).  Abnormal consistency of the gall bladder was observed in females (1/4 treated vs 0/4 controls).  The following microscopic findings were increased (NS) vs 0/4 controls: (i) hyperplasia of the gall bladder in males (2/4) and females (3/4); (ii) bile pigmentation in the gall bladder in males (2/4) and females (2/4); and (iii) chronic inflammation of the gall bladder in females (2/4).  The following clinical chemistry differences (p<=0.05) were noted:  (i) increased phosphate in the >=200 ppm males; (ii) increased bile acids in the >=800 ppm males and 2000 ppm females; (iii) increased triglycerides and decreased albumin in the >=800 ppm animals; (iv) decreased glucose in the 800 ppm females and 2000 ppm animals; (v) decreased total protein and aspartate aminotransferase in the 2000 ppm males; and (vi) increased alkaline phosphatase in the 2000 ppm females.  

      Incidences of thyroid vacuolization were increased (NS; vs 0 controls) in the >=800 ppm females (1-3/4) and the 2000 ppm males (1/4).  Additionally at 2000 ppm, absolute and relative thyroid weights were increased (NS) by 11-39% in both sexes, and incidences of cystic follicles and C-cell hyperplasia were increased (NS) in the thyroid in males (1/4 treated vs 0/4 controls).  However, there were no effects of treatment on levels of T4, T3, or thyroid stimulating hormone.  Kidney pigmentation was increased in the 2000 ppm males (2/4 treated vs 0/4 controls).

      The only findings at 200 ppm were considered adaptive and not adverse.  The increases in N-demethylase and O-demethylase in females (incr. 42-62%) and phosphate and bile acids in males (incr. 8-20%) were considered adaptive because, at this dose, they were not corroborated by gross or microscopic evidence of an adverse effect (such as the liver necrosis noted at 2000 ppm).  Food consumption was decreased only on Day 12 and did not affect body weights or body weight gains at this dose.  Finally, lymphoid hyperplasia was noted in the gall bladder in males, but was only marginally increased (2/4 treated vs 1/4 controls).

      The LOAEL for this study was 800 ppm (equivalent to 27.03/24.99 mg/kg/day in M/F) based on: increased thyroid vacuolization and decreased food consumption and glucose in females; increased platelets, phosphate, bile acids, absolute and relative liver weights, and lymphoid hyperplasia of the gall bladder in males; and decreased albumin and increased triglycerides, N-demethylase, and O-demethylase in both sexes.  The NOAEL is 200 ppm (equivalent to 6.74/6.28 mg/kg/day in M/F).

      This study is classified acceptable/guideline and satisfies the guideline requirement (OPPTS 870.3150; OECD 409) for a 90-day oral toxicity study in the dog.

	870.3200	21/28-Day Dermal Toxicity  -  Rat

      In a 21-day dermal toxicity study (MRID 45121525), MKH 3586 (amicarbazone; 98.5% a.i., Lot/Batch # 05362/0005) was applied undiluted to the shaved intact skin of 10 Sprague-Dawley rats/sex/dose at 0, 200, 500, or 1000 mg/kg bw/day (limit dose) for 6 hours/day. The females received 17 applications and the males received 18 applications over a period of 21 and 22 days, respectively.  Both sexes were dosed for 5 consecutive days during the first two weeks.  During the third week, the females and males were dosed for 7 and 8 consecutive days, respectively, prior to terminal sacrifice.

      No compound-related effects on mortality, clinical signs, body weight, body weight gain, food consumption, ophthalmoscopic examinations, hematology, clinical chemistry, organ weights, or gross or histopathological parameters were observed in either sex.  No treatment-related signs of dermal irritation were observed.
	
	The LOAEL was not observed.  The NOAEL is 1000 mg/kg/day.	

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

	870.3465	90-Day Inhalation  -  Rat

      Not Submitted.

A.4.2	Prenatal Developmental Toxicity

	870.3700a Prenatal Developmental Toxicity Study  -  Rat

      In a developmental toxicity study (MRID 45121530), MKH 3586 Technical (Amicarbazone; 98.1-98.5% a.i.; Lot/Batch # 05362/0005) in 0.5% aqueous carboxymethylcellulose and 0.4% Tween 80 solution was administered daily by oral gavage at a dose volume of 10 mL/kg bw to 30 female Sprague Dawley rats/group at dose levels of 0, 15, 100 or 300  mg/kg/day on gestation days (GD) 6 through 19.  All dams were sacrificed on GD 20; their fetuses were removed by cesarean and examined.  In a supplemental developmental toxicity study (MRID 45121531), conducted to determine whether the skeletal findings observed in the definitive developmental toxicity study at 15  mg/kg/day were incidental and not compound-related,  MKH 3586 Technical (Amicarbazone; 97.8-97.9% a.i.; Lot/batch #05362/0005) in 0.5% (w/v) aqueous carboxymethylcellulose and 0.4% (w/v) Tween 80 solution was administered daily by oral gavage at a dose volume of 10 mL/kg to 30 female Sprague-Dawley rats/group at nominal dose levels of 0, 5 or 15  mg/kg/day on gestation days (GD) 6-19.  All dams were sacrificed on GD 20; their fetuses were removed by cesarean and examined.

      In the main study, hard stools were observed in 9/20 dams at 100 mg/kg/day and 22/27 dams at 300 mg/kg/day beginning several days after dosing began.  At 300 mg/kg/day, clonic convulsions were observed in 3/27 dams.  The death of one pregnant female at 300 mg/kg/day was not clearly treatment-related.  Body weights were dose-dependently decreased (p<=0.05) at 100 (↓7-8%) and 300 (↓4-13%) mg/kg/day on GD 7-20, resulting in decreased (statistics not performed) body weight gains for GD 6-20 both when corrected for (↓45-67%) and uncorrected for (↓19-44%) gravid uterine weights.  Gravid uterine weights were slightly lower than controls (↓14%, not significant), due in part to lower fetal weights.  Food consumption was decreased (p<=0.05) at 100 mg/kg/day on GD 6-9 (↓24-42%) and at 300 mg/kg/day on GD 6-12 and 14-15 (↓15-67%).  Terminal body weights were decreased (p<=0.05) at 100 (↓8%) and 300 (↓13%) mg/kg/day.  The 300 mg/kg/day dams demonstrated increased (p<=0.05) absolute (↑12%) and relative liver weights (incr. 28%).  The maternal LOAEL is 100 mg/kg/day, based on decreased body weights/body weight gains and food consumption, and increased occurrence of hard stools.  The maternal NOAEL is 15 mg/kg/day.

      There were no abortions, premature deliveries, late resorptions or complete litter resorptions.  No effects of treatment were noted on numbers of litters, live fetuses, dead fetuses, early resorptions, placental weight, sex ratio or post-implantation losses.  Decreased (p<=0.01) body weights were observed in the 300 mg/kg/day male and female fetuses (↓13-16%).  There were no treatment-related external, visceral or skeletal variations or malformations.  Multiple dose-related increases (p<=0.05) in retardations of fetal skeletal development were noted.  At >=100 mg/kg/day, incomplete ossification/unossification was observed in:  (i) parietal bones, (ii) interparietal bones; (iii) supraoccipital bones; (iv) squamosal bones; (v) zygoma; (vi) pubis; (vii) xiphoid; and (viii) metacarpals.  Enlargement was observed in the sagittal and squamosal sutures and posterior fontanelle.  Additionally at 300 mg/kg/day, incomplete ossification/unossification was observed in:  (i) nasal bones; (ii) lumbar arches; (iii) sacral arches; (iv) sacral centra; (v) caudal centra; (vi) metatarsals; (vii) manubrium; (viii) sternebrae, segments 2, 3, 4 and 5; (ix) ribs; (x) hyoid body; and (xi) caudal arches.  Enlargement was noted in the frontal, coronal and lamboidal sutures.  No dose-related skeletal findings were noted in the supplemental developmental toxicity study, confirming that skeletal findings observed in the definitive developmental toxicity study at 15 mg/kg/day were probably not compound-related. The developmental LOAEL is 100 mg/kg/day, based on multiple skeletal developmental retardations.  The developmental NOAEL is 15 mg/kg/day.

      This study is classified acceptable/guideline (OPPTS 870.3700a) and satisfies the requirements for a developmental study in the rat.  The Registrant is asked to provide (1) appropriately stained (Alizarin red S and Alcian blue) historical controls for skeletal variations and (2) breeding records for control animals as confirmatory data.

	870.3700b Prenatal Developmental Toxicity Study - Rabbit

      In an oral developmental toxicity study (MRID 45121627), MKH 3586 (Amicarbazone; Lot/batch # 05362/0005; 98.2% a.i.) was administered in 0.5% carboxymethylcellulose in demineralized water via gavage, in a dosing volume of 5 mL/kg, to 22-23 female Himalayan CHBB:HM rabbits/group at dose levels of 0, 5, 20 or 70 mg/kg/day on gestation days (GD) 6 through 28.  All surviving does were sacrificed on GD 29 and their fetuses were removed by cesarean and examined.  Because the control group exhibited an unusually low incidence of malformations, causing the MKH 3586-treated groups to appear to have increased malformation rates by comparison, a supplementary study (MRID 45121626) was conducted with 22 rabbits/group at dose levels of 0 and 70  mg/kg/day to determine whether MKH 3586 caused treatment-related malformations at that dose.

      Maternal toxicity:  At >=20 mg/kg/day in the main study, body weight gains were decreased during treatment (p<=0.05; decr. 29-30%) compared to concurrent controls (GD 6-29).  The decreases during the treatment interval resulted in decreased overall (decr. 10-120%; GD 0-29) body weight gains either uncorrected or corrected for gravid uterine weight, but it is noted that due to a relatively large mean gain during pretreatment (GD 0-6) compared to the other groups, cumulative GD 0-29 gain at 70 mg/kg/day was less than at 20 mg/kg/day (mean body weight at 70 mg/kg/day was about 4% less than controls during treatment, but not significant).  Mean body weights were slightly lower than controls (↓3-4%, not significant) throughout most of treatment.  At 70 mg/kg/day, in the main study, one doe aborted on Day 21.  This animal displayed multiple signs of maternal toxicity (ears cold to touch, reduced feces and urine output, discolored urine and decreased food and water consumption).  Decreased urination in the supplementary study, decreased water consumption in the main and supplementary studies and ears cold to the touch in the supplementary study were observed (in a few animals on GD 6-7 but generally later).  In the supplementary study, body weight gains were decreased (decr. 29%; not significant) during the treatment interval (GD 6-29), resulting in decreased (decr. 23-24%; not significant) overall (GD 0-29) uncorrected or corrected body weight gains.  Body weights also were decreased (p<=0.05) from GD 11-29 (decr. 3-4%).  Maternal food consumption was decreased during GD 6-21 in the main study (p<=0.05; decr. 14-43%) and during GD 6-21 and 24-27 in the supplementary study (decr. 11-48%).  There was an increase in partially necrotic placentas (above historical control range) in the main and supplementary studies (2.2-2.4% fetuses; 11.1-15.0% litters) compared to controls (0.0-0.7% fetuses; 0.0-4.5% litters) and in coarse grained placentas in the main study (8.7% fetuses; 15.0% litters) compared to controls (0%).  No effects on maternal gross pathology were reported.  The maternal toxicity LOAEL is 20 mg/kg/day based on decreased body weight gains during treatment.  The maternal toxicity NOAEL is 5 mg/kg/day.

      Developmental toxicity:  At 70 mg/kg/day, in the main study, a slight, non-significant decrease in fetal weights (↓5-6%) was seen.  There was an increased (p<=0.05) incidence of incomplete ossification of the 5[th] medial phalanx digit, on both the right (39.4% fetuses; 70.0% litters) and left (40.9% fetuses; 70.0% litters) sides.  In the supplementary study, fetal weights were decreased (p<=0.01; decr. 11-13%);  Slightly thickened right 6[th] (2.9% fetuses; 16.7% litters) and left 7[th] (9.4% fetuses; 38.9% litters) ribs and incomplete ossification of the 13[th] caudal vertebra (6.5% fetuses; 38.9% litters) were observed.  There were no effects of treatment the number of resorptions (early, late or complete litter), number of fetuses (live or dead), number of litters, post-implantation loss, fetal weights or sex ratio in either the main or the supplementary studies.   The main study findings were supported by the supplementary study.  The developmental toxicity LOAEL is 70 mg/kg/day based on decreased fetal weights and increased incidences of incomplete ossification of the 5[th] medial phalanx digit (bilateral) and the 13[th] caudal vertebra and slightly thickened ribs.  The developmental toxicity NOAEL is 20 mg/kg/day. 

      This study is classified acceptable/guideline (OPPTS 870.3700b; OECD 414) and satisfies the requirements for a developmental study in the rabbit.

A.4.3	Reproductive Toxicity

	870.3800 Reproduction and Fertility Effects  -  Rat

      In a two-generation reproduction toxicity study (MRID 45121625), MKH 3586 Technical (Amicarbazone; 98.2-99.1% a.i.; Lot/batch #05362/0005) was administered in the diet to 30 Sprague-Dawley rats/sex/dose at nominal dose levels of 0, 100, 500 or 1000 ppm (equivalent to 0/0, 6.4/7.3, 33.9/38.7 or 73.2/84.0 mg/kg bw/day in males/females).  The P and F1 parents were given test diets for 10 weeks before they were mated to produce the F1 and F2 litters.  The F1 pups were weaned at 21 days of age, and approximately 30 pups/sex/group (1 pup/sex/litter as nearly as possible) were randomly selected as parents of the F2 generation.  Sperm parameters were not evaluated in the parental males, and sexual maturation and developmental landmarks were not evaluated in the offspring.

      Parental toxicity:  At 500 ppm, P males and females had reduced cumulative body weight gain during the premating period (males ↓9%, days 0-105 and females ↓20%, days 0-70), although no significant decreases in mean body weight were observed due to higher mean body weights compared to controls.  Food consumption was slightly but statistically significantly reduced only during the first week of treatment.  F1 males and females showed sporadic statistically significant decreases in mean body weight during premating (↓5-20%, males and ↓6%, females).  Cumulative gain decreased 5% in males (days 0-98) and 8% in F1 females (days 0-70), with no decreases in food consumption.  Mean body weights during gestation were reduced in F1 females only (↓6-11%).  Statistically significant reductions in mean body weight of F1 females during lactation (↓6-8%) were not accompanied by reduced gain.  At 1000 ppm, P generation males showed reduced cumulative gain during premating (↓12%, days 0-105); statistically significant reductions in mean body weights were not observed due to higher initial mean body weight compared to controls.  Significantly reduced mean body weights were observed in females during days 0-70 (↓4-6%) and cumulative gain was reduced by 29%.  Significantly decreased body weight in F1 males (↓6-12%, days 0-98) and females (↓10-14%, days 0-70) were observed for the entire premating period; this was due largely to lower initial weights in both sexes, with cumulative gain reduced by 4% in males and 13% in females.  Statistically significant reductions in mean body weights were observed during gestation in both P (↓6-7%) and F1 (↓11-12%) females, along with reduced gain (↓11-12%).  Mean body weights were significantly decreased throughout lactation for P females (↓7-18%) and F1 females (↓10-13%) despite increased food consumption, although cumulative gain was not affected.  Terminal body weights were decreased (p<=0.05) in F1 males and females.  Relative liver weights were slightly increased (p<=0.05) in both P and F1 male and females (↑11-15%), due in part to reduced terminal body weights.  Minimal to slight hepatocytomegaly was noted in F1 males (10/30) compared to controls (0/30), but no microscopic pathology consistent with liver toxicity was observed.  No treatment-related findings were noted at 100 ppm.  The parental toxicity LOAEL is 500 ppm (equivalent to 33.9/38.7 mg/kg bw/day [M/F]), based on decreased body weight/weight gain in both sexes.  The NOAEL is 100 ppm (6.4/7.3 mg/kg bw/day [M/F]).

      Offspring toxicity:  At 500 ppm, decreased body weights were observed on PND 14 and 21 in F1 and F2 pups; cumulative gains for these groups were decreased 10-13% (p<=0.05).  At 1000 ppm, decreased (p<=0.05) body weights were noted on PND 4, 7, 14 and 21 in F1 pups, and on PND 7, 14 and 21 in F2 pups(↓10-23%).  Cumulative gains for this period were decreased 20-25% (p<=0.01).  There were no effects of treatment on the birth, live birth, viability or lactation indices, sex ratio or gross pathology in the F1 or F2 pups.  No treatment-related findings were noted at 100 ppm.  The LOAEL for offspring toxicity is 500 ppm (equivalent to 33.9/38.7 mg/kg bw/day [M/F]), based on decreased pup body weights and overall body weight gains.  The NOAEL is 100 ppm (equivalent to 6.4/7.3 mg/kg bw/day [M/F]).

      Reproductive effects:  The reproductive toxicity LOAEL was not observed.  The reproductive toxicity NOAEL is 1000 ppm (equivalent to 73.2/84.0 mg/kg bw/day [M/F]).

      This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3800; OECD 416) for a three-generation reproduction study in the rat.

A.4.4	Chronic Toxicity

	870.4100a (870.4300) Chronic Toxicity  -  Rat

      In a combined chronic toxicity/carcinogenicity study (MRID 45121512), 50 Fischer 344 rats/sex/dose were exposed to MKH 3586 (Amicarbazone; 98.1-98.6% a.i.; Lot/Batch #: 05362/0005) in the diet at concentrations of 0, 50, 500, or 1250/1000 (M/F) ppm (equivalent to 0/0, 2.3/2.7, 25/30, and 67/65 mg/kg/day in males/females) for up to 24 months.  An additional group of 20 (control and high dose) or 10 (low and intermediate dose) rats/sex/dose were similarly treated and sacrificed at 12 months.

      Mortality, clinical signs, ophthalmoscopic findings, food consumption, hematology, clinical chemistry, urinalysis, and gross and microscopic pathology for both sexes at all doses were unaffected by treatment.  No treatment-related findings were noted at 50 ppm.

      In the interim study at 1250/1000 ppm, decreases (p<=0.05) in body weight were noted throughout most of the 12 months (decr. 5-15%); decreases in body weight gain (calculated by the reviewers) were observed at Weeks 1-13 (decr. 13-25%), 13-50 (decr. 19-27%), and overall (Weeks 1-50, decr. 15-26%); and body weights were decreased (p<=0.05) at the interim sacrifice (decr. 10-16%).  In the main study, decreases (p<=0.05) in body weight were generally observed throughout the 24 months in the 500 ppm females (decr. 3-11%) and in the 1250/1000 ppm groups (decr. 3-18%).  Decreases in body weight gain were observed at >=500 ppm at Weeks 1-13 (decr. 5-24%; both sexes), 13-50 (decr. 26-32%; females only), 50-102 (decr. 43-60%; males only), and overall (Weeks 1-102, decr. 9-23%; both sexes).  Body weights were also decreased (not significant) at the terminal sacrifice in the 1250/1000 ppm groups (decr. 8-9%).

      The LOAEL is 500 ppm (equivalent to 25.3/29.5 mg/kg/day [M/F]) based on decreased body weights in females and body weight gains in both sexes.  The NOAEL is 50 ppm (equivalent to 2.3/2.7 mg/kg/day [M/F]).

      At the doses tested, there was not a treatment related increase in tumor incidence 	when compared to controls.  Dosing was considered adequate, based on decreased body weights in females and body weight gains in both sexes.

      This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4300; OECD 453) for a combined chronic toxicity/carcinogenicity study in rats.

	870.4100b Chronic Toxicity - Dog

      In a chronic oral study (MRID 45121529), MKH 3586 (Amicarbazone technical; 98.1-98.6% a.i., Lot/batch #: 05362/0005) was administered to 4 beagle dogs/sex/dose in the diet at doses of 0, 75, 100, 300 or 1200 ppm (equivalent to 0/0, 1.6/1.8, 2.5/2.3, 8.9/8.7 or 31.5/34.6 mg/kg/day [M/F]) for one year.  In addition to the typical parameters, the investigators evaluated heart rate, blood pressure, electrocardiography (ECG), cortisol, body temperature, circulating thyroid hormones, hepatic enzymes (o-demethylase, n-demethylase and P-450), neurological parameters (mental status/behavior, gait, postural status, postural reactions and spinal/cranial reflex tests) and immunological parameters (cell phenotyping via flow cytometry and immunoglobulin determination). 
      At >=300 ppm, absolute and relative liver weights were dose-dependently increased by 26-41% in males.  O-demethylase activity was dose-dependently increased by 83-158% in males, and cytochrome P-450 activity was increased by 67-73% in females.  Increases in triglycerides (incr. 8-137%) and cholesterol (incr. 16-93%) were observed throughout treatment in both sexes.  Globulin was increased by 18% in females at Day 363.

      Additionally at 1200 ppm, absolute and relative liver weights were increased by 13-16% in the females.  Slight liver hypertrophy and dilatation of the sinusoids were observed in males (1/4 each treated vs 0/4 each controls).  In the liver tissue samples collected at study termination, N-demethylase activity was increased by 30% over controls in males, and O-demethylase activity was increased by 65% in the females.  Cytochrome P-450 activity was increased by 88% in the males.  Albumin was decreased by 9-15% and bile acids increased by 50-100% throughout treatment in both sexes.  Lactate dehydrogenase was increased in males by 179-259%.  Gamma glutamyl transferase was increased by 43% in females at Day 363.  Increased platelets were observed in both sexes (all time points, 18-38% in males and 9-27% in females).  Reductions in % eosinophils in females throughout the study were seen but a clear dose-response was not observed.  Mild abnormal neurological signs were observed in three females at 6 months (abnormal postural reactions) and one female at 12 months (abnormal postural reactions, Hami-standing deficit and lateral hopping deficit).  The study authors concluded that these deficits were not due to direct neurotoxicity but were instead neuromuscular effects possibly secondary to thymic atrophy (see Discussion of this DER for details).  Absolute and relative thymus weights were decreased by 35-40% in the 75 ppm and 1200 ppm males.  These decreases corresponded to the minimal to slight thymic atrophy observed microscopically; however, they were not dose-related or significantly different from the controls.  Thymic atrophy was observed in the 1200 ppm males (from control to high dose, 0/4, 2/4, 1/4, 0/4 and 4/4, p<=0.05 at 1200 ppm) and females (1/4 treated vs 0/4 controls, NS).  The female with persistent neuromuscular effects had thymic atrophy and reduced B cells; findings appeared to be treatment-related.   However, in males thymic atrophy was not dose-related in severity and did not show a strong dose-response for incidence.  Based on findings in females thymic atrophy in males may be treatment related, but a clear dose-response was not observed.  The only findings at 100 ppm were increased O-demethylase activity (incr. 42%) and triglycerides (incr. 43-74%) in males and increased cholesterol in females (incr. 21-29%) but were insufficient to be considered adverse.  Slightly reduced hematocrit and hemoglobin in males at 181 days (decr. 5-8%, p<=0.05) T 100, 300 and 1200 ppm may have been related to treatment but were not considered adverse due to small change and lack of persistence.  There were no effects of treatment on survival, quantitative electroencephalography, body weights/weight gains, food consumption, ophthalmoscope, immunoglobulin quantification, urinalysis or gross pathology.

      The LOAEL is 300 ppm (equivalent to 8.9/8.7 mg/kg/day, M/F), based on effects on the liver, including: increased absolute and relative liver weights, and O-demethylase in males; increased globulin and cytochrome P450 in females; and increased triglycerides and cholesterol in both sexes.  The NOAEL is 100 ppm (equivalent to 2.5/2.3 mg/kg/day, M/F).

      This study is classified as acceptable/guideline and satisfies the guideline requirement (OPPTS 870.4100b, OECD 452) for a chronic oral toxicity study in dogs.

A.4.5	Carcinogenicity

	870.4200a Carcinogenicity Study  -  rat

      See 870.4100 (4300), above.

	870.4200b Carcinogenicity (feeding)  -  Mouse

      In a carcinogenicity study (MRID 45121604), 50 CD-1 (ICR)BR mice/sex/dose were exposed to MKH 3586 (amicarbazone; 98.1-98.6% a.i.; Lot/Batch #: 05362/0005) in the diet at concentrations of 0, 100, 1500, or 4000 ppm nominally (equivalent to 0/0, 15.7/17.9, 244.7/275.0, and 709.0/806.3 mg/kg/day in males/females) for up to 78 weeks.
	
      No adverse treatment-related effects were observed on mortality, food consumption, or gross pathology.  No treatment-related findings were noted at 100 ppm.

      At 4000 ppm, body weights were generally decreased (p<=0.05) at Weeks 3-57 in the males (decr. 3-5%), and at Weeks 53-74 in the females (decr. 5-7%).  This was reflected in decreased body weight gains in males at Weeks 1-13 (decr. 16), and in both sexes at Weeks 1-52 (decr. 14-22) and Weeks 1-78 (decr. 9-13).

      A subclinical treatment-related anemia was observed in the 4000 ppm males.  Decreases (not statistically significant [NS]) in erythrocytes (decr. 11%), hemoglobin (decr. 8%), and hematocrit (45% treated vs 49% controls) were noted at 12 months, and decreases (NS) in erythrocytes (decr. 13%) and hemoglobin (decr. 11%) were noted at 18 months.  At 18 months, decreased (p<=0.05) hematocrit (41% treated vs 47% controls), and increased (p<=0.05) red cell distribution width (15.1% treated vs 13.4% controls) and reticulocytes (4.9% vs 2.6% controls) were observed.  Increased (p<=0.05) hemoglobin distribution width was noted at 12 and 18 months (incr. 12-16%).  Also, hemosiderin pigmentation of the spleen, thought to be hemoglobin-derived, was noted (p<=0.05) in the 1500 ppm males (34%; mild) and the 4000 ppm group (62-66%; moderate).

      The LOAEL is 4000 ppm (equivalent to 709.0/806.3 mg/kg/day [M/F]), based on decreased body weight and body weight gains in males and females, and subclinical anemia and hemosiderin pigmentation of the spleen in males.  The NOAEL is 1500 ppm (equivalent to 244.7/275.0  mg/kg/day [M/F]).

      At the doses tested, there was not a treatment-related increase in tumor incidence when compared to controls.  Dosing was considered adequate based on decreased body weight and body weight gains in males and females, and subclinical anemia and hemosiderin pigmentation of the spleen in males.

      This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4200b; OECD 451) for a carcinogenicity study in mice.
      
A.4.6	Mutagenicity

	Gene Mutation
Guideline 870.5100, bacterial reverse gene mutation assay, Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537
MRID 45121617
Acceptable/guideline
0, 16, 50, 158, 500, 1581 or 5000 ug/mL triazolinon (an intermediate of amicarbazone) in the presence or absence of S9 activation
There was no evidence of induced mutant colonies above background in the presence or absence of S9 activation.
Guideline 870.5100, bacterial reverse gene mutation assay, Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537
MRID 45121616
Acceptable/guideline
0, 16, 50, 158, 500, 1581 or 5000 ug/mL oxadiazolinon (an intermediate of amicarbazone) in the presence or absence of S9 activation
There was no evidence of induced mutant colonies above background in the presence or absence of S9 activation.
Guideline 870.5100, bacterial reverse gene mutation assay, Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537
MRID 45121519
Acceptable/guideline
0, 16, 50, 158, 500, 1581 or 5000 ug/mL technical amicarbazone in the presence or absence of S9 activation
There was no evidence of induced mutant colonies above background in the presence or absence of S9 activation.
Guideline 870.5300, mammalian forward gene mutation assay, V79 Chinese lung fibroblast cells
MRID 45121514
Acceptable/guideline
0, 250, 500, 1000, 2000 or 4000 ug/mL for 5 hours in the presence or absence of S9 activation
There was no evidence of induced mutant colonies above background in the presence or absence of S9 activation.

	Cytogenetics
Guideline 870.5375, in vitro mammalian chromosomal aberration assay (V79 Chinese hamster lung fibroblast cells)
MRID 45121513
Acceptable/guideline
0, 1000, 2000, 3000, 4000 or 5000 ug/mL for 4 hours; harvested at 18 hours post-exposure.  Trial 1 0, 3000, 4000 or 5000 ug/mL; harvested at 18 or 30 hours post-exposure.
There was no evidence of an increased frequency of chromosomal aberration induced above background in the presence or absence of S9 activation.

	Other Genotoxicity
Guideline 870.5395, in vivo mammalian erythrocyte micronucleus test (NMRI mouse)
MRID 45121515
Acceptable/guideline
0 or 100 mg/kg administered via intraperitoneal injection; evaluated at 16, 24 and 48 hours post-dosing.
There was no significant increase in the frequency of micronucleated polychromatic erythrocytes above background in bone marrow at any treatment time.

A.4.7	Neurotoxicity

	870.6100 Delayed Neurotoxicity Study  -  Hen

      Not required.

	870.6200 Acute Neurotoxicity Screening Battery

      In an acute oral neurotoxicity study (MRIDs 45121528), MKH 3586 (amicarbazone; 98.2% a.i., Lot/Batch # 17004/93) in 0.5% methylcellulose-0.4% Tween 80 in deionized water was administered in a single dose by gavage (10 mL/kg) to fasted Fischer 344 rats (12/sex/dose) at doses of 0, 20, 150 or 600 mg/kg in males and 0, 20, 100 or 400 mg/kg in females.  All animals were observed for up to 14 days post-dosing.  Functional observational battery (FOB) and motor activity were evaluated pretreatment and on Days 0 (at the time of peak effect, approximately 3 hours post-dosing), 7 and 14.  At termination, 6 rats/sex/group were perfused in situ for neurohistological examination and tissues from the control and 600/400 mg/kg groups were examined microscopically (brain tissue sections at level 5 from the 150/100 mg/kg groups were also evaluated).  Additionally, two nonguideline acute oral studies were submitted.  The purpose of the first study (MRID 45121526) was to investigate neuropathology (in particular, the time of peak effect and the possible effects on other brain regions at other time points) and to establish if a dose of 400 mg/kg produces changes in quantitative electroencephalography (qEEG) at 8 hrs or 1, 3, 7, 15 or 28 days following acute exposure to seven sets of 6 female rats/dose group at 0 or 400 mg/kg.  Groups were sacrificed at 8 hrs and 1, 3, 7, 15 or 28 days post-dosing for neuropathological evaluation.  The purpose of the second study (MRID 45121527) was to establish a NOAEL for clinical signs and FOB (the most sensitive parameters) in 6 rats/sex/dose group at 0, 2.5 or 10.0 mg/kg, single gavage dose.  Acceptable positive control data (MRIDs 42770301 and 43656301) were previously reviewed.

      At 20 mg/kg, during the FOB, increased incidences (# affected/12 vs. 0 controls) of eyelid ptosis (1-3 of both sexes during home-cage and 7 males in the open-field) and decreased approach response (no reaction in 2-6, both sexes) were observed on Day 0 (approximately 3 hours post-dosing).  Additionally, eyelid ptosis and red nasal stain were noted during daily clinical observations in the males on Day 0.

      At >=150/100 mg/kg, increased incidences of oral stain, red nasal stain and urine stain (females only) were noted as clinical signs beginning on Day 0 and persisting up to one week, except for urine stain (Day 14).  The following treatment-related FOB effects (# affected/12 vs. 0/12 controls, unless otherwise indicated) were observed on Day 0 (approximately 3 hours post-dosing):  home cage, eyelid ptosis in both sexes (9-12); handling, (i) palpebral closure (half-closed to completely closed) in the females (1-12), (ii) ease of removal (minimal resistance with vocalizations to avoiding the hand) in males (1-6), (iii) mild nasal stain (red or clear) in both sexes (2-11), and (iv) mild oral stain (clear or red) in both sexes (2-7); open field, eyelid ptosis in the males (9-12) and in the females (6-11 treated vs 1 control); and reflex/physiologic, decreased approach response (no reaction) in the males (7-11 treated vs 4 controls) and impaired righting reflex (slight incoordination to landing on side) in the females (2-4).  Additionally, decreases (p<=0.05) in mean body temperature were observed in both sexes (34.5-36.4̊C treated vs. 37.0-37.1̊C controls).  Total locomotor activity was dose-dependently decreased on Day 0 in the >=100 mg/kg females (↓30-43%, not statistically significant) and both total motor/locomotor activities reduced in males at 150 mg/kg (↓56-66%, not significant).

      At 600/400 mg/kg, treatment-related mortality was observed in the males (5/12) and females (1/12) on Days 0 or 1 post-dosing.  Increased incidences of the following were noted at the daily clinical evaluation on Day 0 in both sexes: eyelid ptosis (5-6), clear nasal discharge (4-5) and salivation (3-4).  The following treatment-related effects were observed in the FOB evaluation on Day 0 in both sexes (unless otherwise indicated):  home cage, (i) repetitive forepaw movements (1-3), (ii) decreased activity (1-3), (iii) mild tremors (3 males) and (iv) chewing movements (2 males); handling, palpebral closure (half-closed to completely closed) in the males (12) and rigid muscle tone (1-4); open field, (i) mild tremors (3 males), (ii) decreased arousal, some to minimal movement (6 treated females vs 1 control) and (iii) tail mutilation (2 females); and reflex/physiologic, decreased approach response (no reaction) in the females (11 treated vs 4 controls) and impaired righting reflex, slight incoordination to landing on side in males (3 treated vs 1 control).  The only FOB effects that persisted were red nasal stain in one 600 mg/kg male and red oral stain in one 400 mg/kg female on Day 7 and tail lesions (due to self-mutilation) in two 400 mg/kg females on Days 7 and 14.  Lack of motor/locomotor effects in males at 600 mg/kg may have been related to the excessive toxicity/mortality at that dose, based on extreme variability of activities in individual animals of that group.  Slight neuronal necrosis in the midline of the thalamus (reuniens nucleus) was observed in 1/6 male and 1/6 female (both animals survived to Day 14).  No compound-related effects on body weight, brain weight or gross pathology were observed at any dose. 

      The two nonguideline neurotoxicity studies satisfied the purposes for which they were intended.  The first study (MRID 45121526) verified the neuropathological effects observed in the main study high-dose males and females and the time of peak effect at 400 mg/kg.  One female sacrificed 24 hr post-dosing had slight neuronal necrosis of the thalamus in the area of the reuniens nucleus.  Quantitative EEG demonstrated treatment-related electrophysiological changes, primarily increases in total power, absolute delta, theta and alpha, and total power within the theta-beta range at 3, 8 and 24 hr post-dosing (under normotonic, auditory, visual and somatosensory stimulus conditions).  A NOAEL for neurotoxicity was not established in the guideline study; the supplemental study MRID 45121527 established a NOAEL for clinical signs and FOB parameters at 10 mg/kg.

      The neurotoxicity LOAEL is 20 mg/kg based on eyelid ptosis and decreased approach response (both sexes) and red nasal stain (males).  The NOAEL is 10 mg/kg.

      The study is classified as acceptable/guideline in conjunction with MRID 45121527 and satisfies the guideline requirement (OPPTS 870.6200a; OECD 424) for an acute neurotoxicity screening battery in rats.

	870.6200 Subchronic Neurotoxicity Screening Battery

      In a subchronic neurotoxicity study (MRID 45121532), MKH 3586 (amicarbazone; >=98.2% a.i., Lot/Batch # 17004/93) was administered in the diet for 13 weeks to 12 Fischer 344 rats/sex/dose at doses of 0, 100, 500 or 1000 ppm (equivalent to 0/0, 6.7/7.8, 33.4/38.2 or 66.5/75.8 mg/kg/day [M/F]).  Functional observational battery (FOB) and motor activity were evaluated during Weeks -1 (prior to dosing), 4, 8 and 13.  At termination, 6 rats/sex/group were perfused in situ, and tissues from the control and 1000 ppm groups were examined microscopically.  Acceptable positive control data (MRIDs 42770301 and 43656301) were previously reviewed by the Agency.

      No compound-related effects on mortality, clinical signs, FOB, motor or locomotor activity, ophthalmology, brain weight or gross and neuropathology parameters were observed at any dose.  Motor activity data indicated that habituation was unaffected by treatment.

      In the >=500 ppm females, decreases (p<=0.05) were observed in body weights (↓ 6-7%, Weeks 10-13), overall (Day 0-91) body weight gains (calculated by reviewers, decr 17-22%).  Food consumption was reduced (↓ 6-12%) when expressed as g/animal/day but total mean food consumption for the entire study was comparable for all groups on a g/kg bw/day basis (91.0, 90.3, 88.5 and 87.8 g/kg bw/day, controls to high dose).  Body weights, overall body weight gains and food consumption were similar to controls throughout the study in the males.  The LOAEL is 500 ppm (equivalent to 38.2 mg/kg/day [females]) based on decreased body weight and overall body weight gain.  The NOAEL is 100 ppm (equivalent to 7.8 mg/kg/day [females]).  A NOAEL was not established for males.  

	No evidence of neurotoxicity was observed at any dose.

      The submitted study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.6200b) for a subchronic neurotoxicity screening battery in rats.

	870.6300 Developmental Neurotoxicity Study

      In a developmental neurotoxicity study (2001, MRID 45441301), MKH 3586 (97.8-98.4% a.i., batch # 05362/0005) was administered to parent female Wistar rats in the diet at concentrations of 0, 100, 500 or 1000 ppm from gestation day 0 through postnatal day (PND) 21.  The average daily intake of MKH 3586 was approximately 0, 8, 39 and 91 mg/kg/day during gestation and 0, 17, 84, and 177 mg/kg/day during lactation, for the 0, 100, 500, and 1000 ppm groups, respectively.  A Functional Operational Battery (FOB) was performed on 10 dams/dose on gestation days 6, 13, and 20 and on and lactation days 4, 11, and 21.  On postnatal day 4, litters were culled to yield four males and four females.  Offspring, representing at least 20 litters/dose, were allocated for periodic detailed clinical observations including (abbreviated FOB), assessment of motor activity, assessment of auditory startle response habituation, assessment of learning and memory, and neuropathology at study termination (day 75 of age).  Thyroid and neural tissues (10/sex/dose) were assessed histologically on PND 11 and at study termination.  Pup physical development was assessed by bodyweight, and sexual maturation of females (age at vaginal opening) and of males (age at completion of balano-preputial separation) was determined.  
	
      Maternal Toxicity. Treatment-related effects were limited to body weight and gain, increased food consumption and decreased food efficiency.  During gestation, body weight at 1000 ppm was decreased (p<0.05) of 5% on GD 6, with differences persisting to GD 20 (4%, p < 0.05) associated with a 12% decrease (p<0.05) in body weight gain from GD 0 to GD 20.  The high dose group was also associated with increased food consumption (13%) and associated decrease in feed efficiency (22%).   During lactation, maternal body weight was decreased for high-dose rats; decreases averaged 4% on LD 0 to 9% on LD 14 and LD 21 (p<0.05 or 0.01).   Non significantly lower body weights were noted for the 500 ppm dose group of 2-3% but body weight gain during lactation was reduced 28% at 500 ppm and 45% at 1000 ppm.  Food consumption during lactation was increased 7% at 500 ppm and 8% at 1000 ppm.  The combination of body weight decrease and increase food consumption resulted in a decrease in food efficiency or 33% at 500 ppm and 49% at 1000 ppm. The maternal LOAEL is 500 ppm (39 mg/kg/day) based primarily on decreased feed efficiency (combination of decreased body weight gain and increased feed consumption) during lactation.   The maternal NOAEL is 100 ppm (8 mg/kg/day). 
	
      Offspring Toxicity.  Treatment-related effects for offspring were also limited to decreased body weight and body weight gain at 500 ppm and above.  At birth, the average body weight of treated offspring was not different from controls at any dose level.  By PND 11, body weight was decreased 7-8% (p<0.05) for high-dose male and female offspring, this decrease averaged 11-12% (p<0.01) by weaning on PND 21.  Body weight gain was decreased in males and females in the 500 (8-13 %; p<0.05 or p<0.01) and 1000 ppm (11-21 %; p<0.05 or p<0.01) groups.  Offspring in the 500 ppm group had recovered by termination; however, decreased body weight gain persisted to study termination in high-dose animals. The offspring LOAEL is 500 ppm (91 mg/kg/day) based on decreased body weight gain.  The offspring NOAEL is 39 mg/kg/day.

      This study is classified as Acceptable/guideline and satisfies the guideline requirement (OPPTS 870.6300) for a developmental neurotoxicity study in the rat.

A.4.8	Metabolism

	870.7485	Metabolism - Rat

   (1) In a rat metabolism study (MRID 45121701), [triazolinone-3-[14]C] MKH 3586 (amicarbazone; Vial No.: C-710A; radiochemical purity >99%) in water, was administered to 4 male Fischer rats as a single gavage dose at 5.25-5.99 mg/kg.  Samples were collected from these animals to determine elimination kinetics and to quantify metabolites.

      Overall recovery of the radioactive dose was 95% by 72 hours following administration.  The majority of the dose was recovered in the urine within 24 hours (64% dose), indicating substantial absorption.  Fecal excretion accounted for 27% dose within 24 hours post-dose.  Only 2% dose was recovered in the excreta after 24 hours.  At 72 hours following administration, <=1% dose was found in each of the following fractions: carcass and selected tissues, CO2 trap, volatile organic trap, urine trap wash, and cage and feces trap wash.  Concentrations of radioactivity in the selected tissues at 72 hours post-dose were low (0.004-0.073 ug/g).

      HPLC and LC-MS/MS analyses were used to identify the parent and 9 metabolites in excreta from male rats following oral dosing with [triazolinone-3-[14]C] MKH 3586.  Identified compounds in excreta accounted for 74% of the dose.  Unidentified peaks in urine and fecal extracts each accounted for <1% dose, and unanalyzed fractions from urine and feces each accounted for <2% dose.  Overall accountability of the administered dose was ~84%, with minor fractions and unknowns being reported at accounting for "<1% dose".

      Minor amounts of parent were detected in both  urine (~2% dose) and feces (<1% dose).  The major metabolite in excreta was a hydroxylated, deanimated derivative of the parent, iPr-2-OH DA (34% dose), mainly found in the urine (32% dose).  Other hydroxylated deanimated derivatives were also identified: tBu-OH DA (11% dose in urine and 4% dose in feces), and iPr-1,2-diOH DA (6% dose in urine and <1% dose in feces).  The glucuronic acid conjugate of the parent, MKH 3586 GA, accounted for 11% dose, mainly in the feces (10% dose).  Other minor metabolites (each <=3% dose) identified in excreta included:  tBu-1,2-di-iPr-tri-OH DA; tBu-iPr-di-OH DA; iPR-1,3-di-OH DA; tBu-OH-iPr-ene and DA.  Based on the metabolite profile, the metabolism of MKH 3586 in males rats primarily involves deamination followed by hydroxylation with elimination in the urine. Parent also undergoes glucuronic acid conjugation and elimination in the feces.

      This metabolism study in the rat is classified acceptable/guideline and satisfies the guideline requirement for a Tier 1 metabolism study [OPPTS 870.7485, OPP 85-1] in rats. 

   (2) In a rat metabolism study (MRID 45121634), [triazolinone-3-[14]C] 4-methyl MKH 3586 (soil metabolite of amicarbazone; Vial No.: C-803; radiochemical purity >99%) in water, was administered to 4 male Fischer rats as a single gavage dose at 4.86-5.22 mg/kg. Samples were collected from these animals to determine elimination kinetics and to identify and quantify metabolites.

      Overall recovery of the radioactive dose was 91% by 96 hours following administration.  The majority of the dose was recovered in the urine within 12 hours (70% dose), with total urinary excretion accounting for 80% dose, indicating substantial absorption.  Fecal excretion (0-24 hours post-dose) accounted for 8% dose.  Approximately 3% dose was recovered in the excreta after 24 hours.  At 96 hours following administration, 2% dose was found in the urine trap wash and <1% dose was found in each of the following fractions: CO2 trap, volatile organics trap, cage and feces trap wash, carcass and selected tissues.  At 96 hours post-dose, the concentration of radioactivity in the tissues was low (0.002-0.028 ug/g), with <0.1% dose in each tissue.

      HPLC, [1]H-NMR, and LC-MS/MS analyses identified 12 components in excreta from rats following oral dosing with [triazolinone-3-[14]C] 4-methyl MKH 3586.  Four of these compounds were present as chiral pairs.  Identified compounds accounted for 75% of the dose.  All unidentified peaks each accounted for <=1% of the dose, and individual unanalyzed fractions each accounted for <2% dose.  The overall accountability of the administered dose was ~82%.  The proposed pathway for biotransformation of [triazolinone-3-[14]C] 4-methyl MKH 3586 in rats is presented in the Appendix to this DER.

      The test substance was not detected in the urine or feces.  Hydroxylation of the soil metabolite at the isopropyl moiety formed the major metabolite, 4-Me-i-Pr-2-OH DA MKH 3586 (29% dose), which was found primarily in urine (28% dose).  Further hydroxylation at the tertiary butyl group resulted in the formation of 4-Me-t-Bu-iPr-2-di-OH DA MKH 3586, which accounted for 12% dose.  Alternatively, additional hydroxylation of the isopropyl moiety formed 4-Me-iPr-1,2-di-OH DA MKH 3586, which constituted 9% dose in urine.  Desmethylation of the parent formed DA MKH 3586 (<1% dose), which was then hydroxylated at the isopropyl moiety to form iPr-2-OH DA MKH 3586 (8% dose).  The parent was also be hydroxylated at the tertiary butyl moiety to form 4-Me-tBu-OH DA MKH 3586 (9% dose), which was only detected urine.  The other minor identified metabolites occurred resulted from further oxidation, usually hydroxylation.  The pathway for the metabolism of 4-methyl DA MKH 3586 in rats primarily involves a series of hydroxylation reaction.  No conjugates were detected.

      This metabolism study in the rat is classified acceptable/guideline and satisfies the guideline requirement for a Tier 1 metabolism study [OPPTS 870.7485, OPP 85-1] in rats.
	870.7600	Dermal Absorption - Rat

      Not Submitted.

A.4.9	Immunotoxicity

	870.7800	Immunotoxicity

      In an immunotoxicity study (MRID 48407504) Amicarbazone (99.3% a.i. batch 0709AMZ120-1) was administered to 10 female Fischer (F344/DuCRL [CDF[(R)]]) rats/dose via diet at dose levels of 0, 250, 1000, or 2500 ppm (0, 20, 81, 195 mg/kg/day) for 28 days.  The positive control group consisted of 10 female Fischer (F344/DuCRL [CDF[(R)]]) rats were administered 50 mg/kg cyclophosphamide via intraperitoneal injection on Days 24-27.  On Day 24 of the study, all animals were immunized via 0.5 mL intravenous injection with 2.0x10[8] sheep red blood cells (SRBCs).  On Day 28, T-cell dependent antibody response (TDAR) was determined with an antibody forming cell (AFC) assay.  Parameters evaluated include mortality, clinical signs of toxicity, body weight, body weight gain, food consumption, water consumption, organ weights (i.e. spleen and thymus), macroscopic pathology, and assay-specific parameters.

In the treated animals there were no unscheduled mortalities; there were no treatment-related effects on body weight, water consumption, or thymus weight; and there were no treatment-related findings for clinical signs of toxicity or macroscopic pathology.  At the 2500 ppm dose, the treatment-related effects were decreased body weight, food consumption, and absolute spleen weight.

      The systemic toxicity NOAEL is 1000 ppm (81 mg/kg/day) and the LOAEL is 2500 ppm (195 mg/kg/day) based on decreased body weight gain, food consumption, and absolute spleen weight.
      
      The antibody forming cell (AFC) assay results showed that there was decrease in the quantity of spleen cells/spleen in the 2500 ppm dose group with no statistical differences in the quantity of IgM AFC/spleen cells or IgM AFC/spleen in any treatment group when compared with the vehicle controls.  A high inter-individual variability was noted in all the treatment groups as well as in the control group.  Evaluation of individual animal data showed a distribution demonstrating significant suppression of SRBC antibody response at the 2500 ppm dose.

The Natural Killer (NK) cells activity assay was not performed in this study.  Under HED guidance, NK cells activity assay is not required due to a positive response in the TDAR study (i.e. AFC assay).

The immunotoxicity NOAEL is 1000 ppm (81 mg/kg/day) and the LOAEL is 2500 ppm 195 mg/kg/day) based on results of an AFC assay.

      This immunotoxicity study in rats is classified as Acceptable/Guideline and satisfies the guideline requirement for an immunotoxicity study (OPPTS 870.7800).

A.4.9	Special/Other Studies

Nonguideline Mechanistic Studies

Subchronic Mechanistic Feeding Study in Rats

      In a nonguideline mechanistic subchronic oral toxicity study (MRID 45121603), MKH 3586 (Amicarbazone; >=98% a.i., Lot/batch # PT:05362/0005) was administered to 25 Fisher 344 rats/sex/group in the diet at dose levels of 0, 50, 1250 or 2500 ppm (equivalent to 0/0, 0.8/0.6, 19.4/13.5 or 40.0/28.8 mg/kg bw/day in males/females) for 10 weeks.  Additionally, 5 rats/sex/group were fed test diets containing 0 and 2500 ppm for 10 weeks followed by a four-week recovery period.  The purpose of this study was to determine whether thyroid hormone changes (observed in subchronic and chronic toxicity studies with MKH 3586 in the rat) are due to a direct effect of the test substance on the thyroid or to effects on liver enzymes that affect thyroid hormone metabolism.  Thyroid function was assessed using the perchlorate discharge assay and by determining circulating levels of thyroid hormones T3 and T4 (total and free measured for both) and TSH.  Liver function was assessed by determining liver weight and levels of hepatic UDP-glucuronosyltransferase and 5'deiodinase activities.  Additionally, estrous cycle duration and frequency were examined.

      In the 1250 ppm females, TSH, free T3, T3, free T4 and T4 were increased by 27-61%.  In the 2500 ppm females, levels of these thyroid hormones were increased over controls by 5-24% (not significant [NS]) but to a lesser extent than at 1250 ppm.  Increased T3 (incr. 52%) and free T3 (incr. 31%; NS) were observed in males at 2500 ppm.  At the end of the recovery period, levels of TSH, T3 and T4 in 2500 ppm males and females were comparable to controls.  In the perchlorate assay, thyroid to blood ratio of [125]I in MKH 3586 treated groups were comparable to negative controls in males and females, indicating that the increase in thyroid hormones is not due to increased synthesis.  Thus, an organ or tissue other than the thyroid must be responsible for altered levels of thyroid hormones in rats treated with >=1250 ppm test diets.  There were no treatment-related effects on thyroid weight or microscopic changes in the thyroid at any dose.

      In the liver, UDP-GT activity was increased by 109-241% in the >=1250 ppm males and females and remained increased by 27% in the 2500 ppm females at the end of recovery.  Absolute/relative (to body) liver weights were increased in the main study 1250 ppm males (incr. 9/14%) and females (incr. 5/12%) and in the 2500 ppm males (incr. 25/32%) and females (incr. 18/31%).  Relative liver weights remained increased (incr. 7%) in the 2500 ppm males at the end of the recovery period.  Absolute liver weights in males and absolute and relative liver weights in females were comparable to controls at the end of the recovery period.  Because metabolism of the test substance is primarily via glucuronidation by UDP-GT, it is postulated that MKH 3586 competitively inhibited UDP-GT glucuronidation of T3 and T4 at 1250 ppm, resulting in increased levels of these thyroid hormones in the blood serum.  At 2500 ppm, further induction of UDP-GT began to compensate for this competitive inhibition, allowing T3 and T4 levels to decline.  

      In the females, terminal body weights were decreased by 7-10% at >=1250 ppm during the main study and remained decreased by 8% at 2500 ppm at the end of recovery.  An increase in the number of animals spilling food was noted at 2500 ppm.  Food consumption was intermittently decreased on a g/animal/day basis by 3-16% (p<=0.05) in the: (i) main study and recovery group males at 2500 ppm during treatment; (ii) main study and recovery group females at >=1250 ppm during treatment; and (iii) 2500 ppm females at the end of the recovery period.  On a g/kg bw/day basis, food consumption was similar among groups.  Absolute/relative uterine weights were decreased at 1250 ppm (decr. 15/9%) and 2500 ppm (decr. 24/15%).  Decreases in absolute (decr. 20%) and relative (decr. 13%; NS) uterine weights continued in the 2500 ppm recovery group animals.  There were no effects of treatment on the number of estrous cycles.  However, a minor increase of 4% in the duration of the estrous cycle was observed at 2500 ppm.

      In conclusion, thyroid hormones were increased in the >=1200 ppm females and 2500 ppm males.  However, thyroid to blood ratios of [125]I in treated groups were comparable to negative controls, indicating there was no impairment thyroid hormone synthesis.  Thus, the differences in thyroid hormones must be due to metabolism at an extra-thyroidal site.  The liver was implicated as this site based on increased liver weights and UDP-glucuronosyltransferase activity.

	The submitted special mechanistic subchronic oral toxicity study in the rat is classified as 	acceptable/nonguideline and satisfies the purpose for which it was intended.

In vitro Studies on Enzymes of Thyroid Hormone Regulation.  MRID 45121618.

      In a nonguideline study (MRID 45121618), possible interactions of MKH 3586 (Amicarbazone; 98.2% a.i., Lot/batch # 05362/0005, TOX 4490), MKH 3594 (N-desamino metabolite; 99.4% a.i., batch # 200898) and KOK 9422 (putative hydrolysis metabolite; 100% a.i., batch # 130898) with the enzymes involved in the synthesis of thyroid hormones and the regulation of the hypothalmus-pituitary-thyroid axis were investigated using three in vitro systems: (i) thyroid peroxidase (TPO), the key enzyme responsible for organification of iodide and the coupling of iodinated tyrosine residues to both T3 and T4; (ii) iodothyronine deiodinase type I (ID-I), catalyzing the phenolic ring deiodination of T4 to form T3 in peripheral organs like thyroid, liver and kidney; and (iii) iodothyronine deiodinase type II (ID-II), catalyzing the phenolic ring deiodination of T4 to T3 in the hypothalamus and pituitary gland.

      Positive controls adequately demonstrated the sensitivity of each of these in vitro assays through strong, concentration-dependent inhibition of the enzyme of concern.  Amitrole inhibited TPO-catalyzed guaiacol oxidation and iodine formation.  Ethylenethiourea (ETU) temporarily inhibited iodine formation, not by inhibiting TPO itself but instead by reducing iodinating species (i.e., trapping).  Propylthiouracil (PTU) inhibited ID-I, and iopanoic acid inhibited ID-II.

      Neither MKH 3586, MKH 3594, nor KOK 9422 inhibited TPO, ID-I or ID-II at concentrations up to 1 mM, suggesting that MKH 3586 does not affect the iodide organification step of thyroid hormone synthesis (via either inhibition of TPO or trapping of iodine) or the peripheral metabolism of thyroid hormones via Type I or Type II deiodinases in vivo.  These findings support the conclusion that MKH 3586 does not affect the major enzymes involved in the synthesis or regulation of thyroid hormones and are consistent with the findings of the nonguideline subchronic mechanistic study in rats (MRID 45121603) which indicate that upregulation of UDP-glucuronosyl transferase in the liver may account for alterations in the thyroid hormone profile.

      The submitted special in vitro mechanistic study is classified as acceptable/nonguideline and satisfies the purpose for which it was intended, to investigate the possible inhibition of enzyme regulation of thyroid hormone synthesis and metabolism.

Nonguideline Behavioral Study in Rats.  MRID 45121511, 45121521, 45121629.

      In three special studies (MRIDs 45121511, 45121521 and 45121629),  BAY 31-4666 (amicarbazone, 98.4% a.i., Batch #: 05362/0005) was administered in a single gavage dose in 0.5% aqueous tylose suspension (w/v) plus 0.4% TWEEN 80 (5 mL/kg) to 6-10 male HsdCbp WU rats/dose at 0, 1.0, 2.5, 5, 10, 20 or 100 mg/kg bw.  The purpose of the studies was to assess central nervous system (CNS) effects in rats dosed with BAY 31-4666 and to determine a LOAEL.  In the first two studies, rats were dosed with 0, 1,0, 20.0 or 100.0 mg/kg BAY 31-4666.  In (1) MRID 45121529, 6 rats/dose were evaluated for behavioral or physiological changes by the modified Irwin test every 15 minutes for 3 hrs post-dosing, then at 24 hrs post-dosing.  In (2) MRID 45121521, 5 rats/dose were evaluated for catalepsy (5 rats/dose) and 6 rats/dose for body temperature (both assessed every 30 minutes post-dosing up to 240 minutes; pre-dosing body temperature measurements also taken), and 10 rats/dose were evaluated for psychomotoric activity (traveling distance, resting time and rearing) at 30, 60 and 120 minutes post-dosing.  In the third study (MRID 45121511), 6 rats/dose were administered BAY 31-4666 at 0, 2.5, 5.0 or 10.0 mg/kg and evaluated by the modified Irwin test every 15 minutes for 3 hrs post-dosing, then at 24 hrs post-dosing. 

      No treatment-related effect was observed in the 1 mg/kg group, nor on clinical signs at 2.5 and 5 mg/kg.  No treatment-related effect was observed during the catalepsy test.  A treatment-related decrease in body temperature was observed in the 100 mg/kg males; however, the minor decreases (<=0.6̊C) were considered not to be biologically adverse.  

      The following clinical signs (# rats) were observed: (i) sedation at 10 (1), 20 (3) and 100 (6) mg/kg; (ii) ptosis at 10 (1), 20 (3) and 100 (6) mg/kg; and (iii) salivation at 10 (2), 20 (2) and 100 (6).  Additionally, piloerection (2), Straub phenomenon (4), and prone position (2) were observed in the 100 mg/kg group.  The effects were first observed at 30 minutes post-dose, and no effect was observed in any animal after 150 minutes post-dose, with the higher dose groups showing greater persistence of effects.

      A dose- and time-dependent effect was demonstrated on motor activity.  In the 20 and 100 mg/kg groups, decreased (↓17-28%; p<=0.05) distance traveled and increased (↑19-36%; p<=0.05) resting time was observed at 30 minutes post-dose.  Rearing was decreased (↓52-54%; p<=0.05) at 100 mg/kg at 30 and 60 minutes post-dose; a non-significant reduction of ↓34% was also observed at 20 mg/kg at 60 minutes.  The CNS effects LOAEL is 10 mg/kg, based on transient sedation, ptosis and salivation.  The NOAEL is 5 mg/kg. 

      This study is classified as acceptable/nonguideline and satisfies the purpose for which it was intended. 

Nonguideline Study of Central Nervous System Safety Pharmacology in Mice

      In a nonguideline study (MRID 45121522), a single dose of MKH 3586 (Amicarbazone; Lot/batch # 05362/0005; 98.4% a.i.), in a 0.5% aqueous tylose suspension (w/v) plus 0.4% Tween-80, was administered orally, in a dosing volume of 10 mL/kg, to 40 male HsdWin: NMRI mice/group at dose levels of 0, 1, 20 or 100 mg/kg/day.  Control animals received an appropriate volume of vehicle without test substance.  The following tests were performed (10 animals/test): (1) hot plate test (analgesia), 56̊C at pretreatment, 30, 45, 60 and 120 min post-dosing; (2) traction, balance rod and electroshock test (reduction of traction force, sensorimotor disturbances and anticonvulsive activity at 30, 40 and 50 minutes post-dosing, respectively); (3) pentylenetetrazole test (pro- and anticonvulsive activity at 45 minutes post-dosing; volume of pentylenetetrazole required to induce clonic seizure) and (4) hexobarbital test (potentiation or reduction of narcotic effects at 60 minutes post-dosing, duration of anesthesia measured).  The objective of this study was to assess the effects of a single oral administration of MKH 3586 on the central nervous system of mice.

      At 100 mg/kg, mice showed increased (not significant unless noted) response times compared to controls to a nociceptive stimulus at 30 (incr. 24%), 45 (incr. 45%), 60 (p<=0.05; incr. 67%) and 120 (incr. 32%) minutes after dosing.  2/10 mice were observed to have reduced traction force and impaired motor coordination, compared to 0/10 in the controls and in the 1 and 20 mg/kg groups.  An increased (p<=0.05) mean threshold dose of pentylenetetrazole was noted (incr. 16%) compared to controls, indicating an anticonvulsant effect by the test compound.  Mice also were observed to exhibit sedation and partial ptosis approximately 20 minutes after treatment.

      Following treatment with the test compound, all mice at all doses exhibited tonic convulsions in the electroshock test.  Additionally, no treatment-related effects were observed on the duration of hexobarbital-induced anesthesia.  There were no effects on the CNS of the 1 and 20 mg/kg mice in any test.

      The data indicate that a single oral dose of MKH 3586 at 100 mg/kg causes minimal CNS functional impairment, characterized by increased reaction times to nociceptive stimuli, reduced traction force, impaired motor coordination, sedation, partial ptosis and a mild anticonvulsive effect.

      The submitted study is classified as acceptable/nonguideline and satisfies the intended purpose of assessing the effects of a single oral administration of MKH 3586 on the central nervous system of mice.

Appendix B.  Physical/Chemical Properties and Structures.

Table B1.  Amicarbazone Physico-Chemical Properties
Parameter
                                     Value
                                   Reference
Molecular Weight
                          [C10H19N5O2] = 241.29 g/mol
                                      N/A
pH
                              7.06 (2.5% slurry)
                                 MRID 45121501
Water solubility (20°C)
                                    4.6 g/L
                                 MRID 45121501
Solvent solubility (20°C to 25°C)
                       n-Heptane = 0.07, xylene = 9.2, 
                      1-octanol = 43,  2-propanol = 110,
                     ethyl acetate = 140, acetone >250,
polyethylene glycol = 79, acetonitrile >250, dimethylsulfoxide = 250, dichloromethane >250
                                 MRID 45121502
Vapor pressure (25°C)
                   3.00 x 10[-6]Pa @ 25C (=2.3x10[-8] mmHg)
                             1.30 x 10[-6]Pa @ 20C
                                 MRID 45121501
Dissociation constant, pKa
              Does not dissociate. No acidic or basic properties.
                                 MRID 45121501
Octanol/water partition coefficient, log KOW (20°C)
                          log Pow =1.23 @ pH 7 (20C)
                                 MRID 45121502
Soil Half-life (or other relevant information from EFED Drinking water assessment)
                    Aerobic Soil Metabolism t1/2 = 87 days
                            DP Barcode No. D384516

Table B2.  Chemical Structures of Residues of Concern for Tolerance Enforcement Purposes:  Amicarbazone, Desamino amicarbazone and iPr-2-OH desamino amicarbazone.
Amicarbazone

4-amino-N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide
Desamino amicarbazone (metabolite proposed for regulation)

N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide
iPr-2-OH desamino amicarbazone 

N-(1,1-dimethylethyl)-4,5-dihydro-3-(1-hydroxy-1-methylethyl)-5-oxo-1H-1,2,4-triazole-1-carboxamide
Appendix C.  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.  These data, which include studies from the Pesticide Handlers Exposure Database Version 1.1 (PHED 1.1); the Agricultural Handler Exposure Task Force (AHETF) database; and the Outdoor Residential Exposure Task Force (ORETF) database; are subject to ethics review pursuant to 40 CFR 26, have received that review, and are compliant with applicable ethics requirements.  For certain studies that review may have included review by the Human Studies Review Board.  Descriptions of data sources as well as guidance on their use can be found at http://www.epa.gov/pesticides/science/handler-exposure-data.html and http://www.epa.gov/pesticides/science/post-app-exposure-data.html.