

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

              OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION


MEMORANDUM			

February 23, 2011			
                                                
SUBJECT:	Acibenzolar-S-methyl Human Health Risk Assessment for Proposed Use of Acibenzolar-S-methyl on Low Growing Berries Crop Subgroup 13-07G.

PC Code:  061402
DP Barcode: 387071
Decision No.:  443561
Registration No.: 100-RGAG
Petition No.:  7E7818
Regulatory Action: Section 3 Registration 
Risk Assessment Type:  Single Chemical Aggregate
Case No.:  7031
TXR No.:  NA
CAS No.:  135158-54-2
MRID No.:  NA
40 CFR:  180.561

FROM:	Becky Daiss, Biologist 
            James Scott Miller, Environmental Scientist
            Thurston Morton, Chemist
            Abdallah Khasawinah, Toxicologist
            Risk Assessment Branch 4
            Health Effects Division (7509P)

THROUGH:	Elissa Reaves, PhD, Chief
      Susan V. Hummel, Branch Senior Scientist
            Risk Assessment Branch 4, HED (7509P)
            
TO:		Sydney Jackson, Risk Manager Review
		Risk Integration, Minor Use & Emergency Response Branch
		Registration Division (7505P)
            
	This document provides the Health Effects Division's (HED's) risk assessment of use of acibenzolar-S-methyl on low growing berries, crop subgroup 13-07G.  









TABLE OF CONTENTS				
											pg.
1.0 	EXECUTIVE SUMMARY	4

2.0	HED RECOMMENDATIONS	6
2.1	Data Deficiencies/Conditions of Registration	6
2.2	Tolerance Considerations	6
	2.2.1 	Enforcement Analytical Method	6
	2.2.2 	International Harmonization	6
	2.2.3 	Recommended Tolerances	6
	2.2.4 	Revisions to Petitioned-For Tolerances	7
	
3.0	INGREDIENT PROFILE	7
3.1	Chemical Identity 	7
3.2	Physical/Chemical Properties	8
3.3	Pesticide Use Pattern	8
	3.3.1 	Registered Products	8
	3.3.2 	Registered and Proposed New Uses	8

3.4	Anticipated Exposure Pathways	9
3.5	Consideration of Environmental Justice	9

4.0	HAZARD CHARACTERIZATION/ASSESSMENT	9
4.1	Toxicology Studies Available for Analysis	9
4.2 	Absorption, Distribution, Metabolism, & Elimination	9
4.3	Toxicological Effects	10
4.4	Safety Factor for Infants and Children	11
	4.4.1 	Completeness of the Toxicology Database	12
      4.4.2 	Evidence of Neurotoxicity	12
      4.4.3 	Evidence of Sensitivity/susceptibility in the Developing or Young	12
      4.4.4 	Residual Uncertainty in the Exposure Data Base	13
4.5	Toxicity Endpoint and Point of Departure Selections	13
	4.5.1 	Dose-Response Assessment	13
      4.5.2 	Recommendation for Combining Exposure Routes	14
      4.5.3	Cancer Classification 	14
      4.5.4 	Summary of Points of Departure Used in Risk Assessment	15

5.0 	DIETARY AND DRINKING WATER RISK ASSESSMENT	16
5.1	Metabolite/Degradate Residue Profile	16
	5.1.1 	Summary of Plant and Animal Metabolism Studies	16
      5.1.2 	Comparison of Metabolic Pathways	16
      5.1.3 	Environmental Fate and Transport	16
      5.1.4 	Residues of Concern Summary and Rationale	17
5.2	Food Residue Profile	18
      5.2.1	Residues in Crops, Livestock and Poultry	18
5.3	Water Residue Profile	18
	5.3.1 	Estimated Drinking Water Concentrations	18
5.4	Dietary and Drinking Water Exposure and Risk	19
	5.4.1 	Acute Dietary Exposure and Risk	19
      5.4.2 	Chronic Dietary Exposure and Risk	20

6.0 	RESIDENTIAL EXPOSURE AND RISK	20
6.1 	Residential Bystander Postapplication Inhalation Exposure	20
6.2 	Spray Drift	21

7.0 	AGGREGATE EXPOSURE AND RISK ASSESSMENT	21
7.1 	Short-Term Aggregate Risk to Residential Applicators	22

8.0	CUMULATIVE RISK	22

9.0	OCCUPATIONAL EXPOSURE AND RISK	23
9.1	Exposure Scenarios	23
9.2	Handler Exposure	23
	9.2.1 	Handler Exposure Scenarios	23
      9.2.2 	Handler Exposure Data	23
      9.2.3 	Handler Exposure Assumptions	23
      9.2.4 	Handler Exposure and Risk Estimates	24
9.3	Post Application Exposure	25
	9.3.1 	Post Application Exposure Scenarios	25
      9.3.2 	Post Application Exposure Assumptions	25
      9.3.3 	Post Application Exposure and Risk Estimates	26
      9.3.4 	Restricted Entry Interval	26
10.0	REFERENCES	26

Appendix A. Toxicity Profile and Executive Summaries	28
	A.1  	Toxicity Data Requirements	28
	A.2  	Toxicity Profiles	29
	A.3 	Hazard Identification and Endpoint Selection	29
	A.4  	Executive Summaries	39
Appendix B. Metabolism 	45
Appendix C. Physical/Chemical Properties	47
Appendix D. Review of Human Research	47



1.0 	EXECUTIVE SUMMARY

      This assessment provides information to support an amended Section 3 registration for the use of acibenzolar-S-methyl on low growing berries, crop subgroup 13-07G.  This document addresses the exposures and risks associated with exposures from currently registered uses and the proposed new uses of acibenzolar-S-methyl.  It also assesses potential enhanced sensitivity of infants and children from dietary and/or residential exposure as required under the Food Quality Protection Act (FQPA) of 1996.
        
Use Profile

	Acibenzolar-S-methyl is a plant activator.  It is a member of the benzothiadiazole class of compounds.  Acibenzolar-S-methyl is registered to Syngenta Crop Protection, Inc for use on a number of leafy and fruiting vegetables crops and tobacco for the control or suppression of downy mildew, black rot, white rust, bacterial leaf spot and speck, and blue mold.  Permanent tolerances ranging from 0.1-3.0 ppm are established under §180.561(a).  Acibenzolar-S-methyl is also used in commercial seed treatment operations for protection against certain early season seed and seed-borne nematodes in cotton and on sod farms, golf courses, collegiate athletic fields, and lawns around commercial and industrial buildings to control turf diseases.  Acibenzolar-S-methyl acts to protect plants against invading pathogens by inducing a natural defense reaction that enhances the plants ability to ward off disease.  Agricultural applications may be made using ground or aerial equipment.  Acibenzolar-S-methyl is also registered for residential application to turf grass using handheld spray equipment.  
   
Proposed New Uses

	The Interregional Research Project No. 4 (IR-4), on behalf of the Agricultural Experiment Stations of CO, NM, UT, ID, and WA, is requesting to amend the product label for Actigard(R) (EPA Reg. No. 100-922), a water-dispersible granule (WG) formulation containing 50% acibenzolar-S-methyl to add a new use on the low growing berry crop subgroup 13-07G.  Actigard(R) is proposed for multiple foliar applications on the low growing berry subgroup using ground or aerial equipment at a maximum of 0.023 lb ai/A per application, a maximum seasonal rate of 0.188 lb ai/A, and a 0-day pre-harvest interval (PHI).  

Hazard Identification
      
      The toxicology database for acibenzolar-S-methyl is adequate for evaluating and characterizing toxicity and selecting endpoints for purposes of this risk assessment.  

	In subchronic and chronic oral studies in rats, dogs and mice, signs of mild regenerative hemolytic anemia were consistently observed in all three species.  Additional toxic effects observed in these studies included decreases in body weight, body weight gain and/or food consumption.  No other significant treatment-related effects of toxicological concern were observed in these subchronic and chronic oral studies.  No neurotoxic effects were seen at the highest dose tested in a subchronic neurotoxicity study in rats.  In a 28 day dermal toxicity study in rats no systemic or dermal effects were seen at the limit dose.  
		In a developmental toxicity and developmental neurotoxicity studies in rats, treatment related effects were observed in fetuses at levels that were not toxic to the parent indicating increased sensitivity of rat fetuses compared to adults.  Increased sensitivity was not observed in a developmental toxicity study in rabbits, or in 1- and 2-generation reproduction studies in rats.  In a 28 day dermal developmental toxicity study in rats, no maternal or developmental toxicity was observed at dose levels up to 500 mg/kg/day, the highest dose level tested.  

	Acibenzolar-S-methyl was classified by HED's Hazard Identification Assessment Review Committee (HIARC) as "not likely" to be a human carcinogen based on negative carcinogenicity findings in male and female rats and mice and negative results in an acceptable battery of genotoxicity studies.  

	Acibenzolar-S-methyl showed no significant toxicity in a battery of acute toxicity tests (Toxicity Category III or IV in all tests) but showed considerable skin-sensitivity.  The end-use product (ActigardTM 50Wg) displayed no significant acute toxicity and no dermal sensitizing potential.

      An immunotoxicity study required as part of new 40 CFR Part 158 data requirements for registration of a pesticide has been submitted and is being reviewed.  Based on a preliminary review, the study is acceptable and indicates no evidence of immunotoxicity.  
      
Dose Response Assessment

      Toxicological endpoints were selected for dietary/drinking water, occupational and residential exposure scenarios.  Acute and chronic reference doses (RfDs) were selected for assessment of food and drinking water exposures.  An acute RfD for the general population was not selected because no effect attributable to a single day oral exposure was observed in animal studies.  Acute and chronic RfD's for females 13-49 were selected from a developmental neurotoxicity toxicity study in rats based on changes in brain morphometrics in the cerebellum.  A chronic RfD for the general population was selected on the basis of hemolytic effects seen in a chronic feeding study in dogs and other co-critical toxicity studies. Toxicological endpoints for inhalation, dermal, and incidental oral exposures were selected from the developmental neurotoxicity study in rats.  A dermal absorption factor was selected from a dermal absorption study in rats.  An uncertainty factor of 100X was applied to endpoints selected for all exposure routes (10x for interspecies extrapolation, 10x for intraspecies variation).   
      
Exposure/Risk Assessment and Risk Characterization

	Risk assessments were conducted for dietary (food and water) and occupational exposure pathways based on the request for new uses of acibenzolar-S-methyl on low growing berries.   A new residential assessment is not required because the proposed new use does not include residential applications or exposures.  Refined acute and screening level chronic dietary and drinking water risk assessments for acibenzolar-S-methyl showed that dietary and drinking water exposure estimates are below HED's level of concern for the general population and all population subgroups.  Occupational exposure and risk estimates indicate that worker handler and post-application exposures are not of concern at the maximum allowable application rates for the proposed new uses.  Aggregate risks are not of concern.

Use of Human Studies

	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 studies, listed in Appendix D, have been determined to require a review of their ethical conduct.  Some of these studies are also subject to review by the Human Studies Review Board.  All of the studies used have received the appropriate review.

2.0       HED RECOMMENDATIONS

2.1	Data Deficiencies/Conditions of Registration 

      Submission of a reference standard for the metabolite CGA 210007, and a revised Section F are required.  An immunotoxicity study required as part of new 40 CFR Part 158 data requirements for registration of a pesticide has been submitted and is being reviewed.   Based on a preliminary review, the study is acceptable and indicates no evidence of immunotoxicity.

2.2	Tolerance Considerations

      2.2.1	Enforcement Analytical Method

      HPLC/UV Method AG-617A is available for tolerance enforcement.  This method has undergone a successful tolerance method validation by the Analytical Chemistry Branch of BEAD.  The method was forwarded to FDA for inclusion in PAM Volume II.  Briefly, the method consists of an initial hydrolysis with NaOH to convert acibenzolar-S-methyl to CGA-210007 followed by methanol extraction.  Residues are then diluted with HCl and purified by a series of solid-phase extraction steps.  Prior to HPLC/UV analysis, residues are partitioned into ethyl acetate, dried down, and re-dissolved in phosphoric acid.  This method has a limit of quantitation (LOQ) of 0.02 ppm.  The method includes optional detection via HPLC/MS, giving a means of residue confirmation.

      2.2.2	International Harmonization
      
      There are no Codex, Mexican, or Canadian maximum residue limits for acibenzolar-S-methyl in/on any commodity.  Therefore, international harmonization is not an issue. 
      
      2.2.3	Recommended Tolerances
      
      HED has examined the residue chemistry database for acibenzolar-S-methyl.  Pending submission of a revised Section F (see requirements under Proposed Tolerances), there are no residue chemistry issues that would preclude granting a Section 3 registration for the requested use of acibenzolar-S-methyl on crop subgroup 13-07G, or the establishment of a tolerance for residues of acibenzolar-S-methyl, as follows:
Table 1.	Tolerance Summary for Acibenzolar-S-methyl.
Commodity
                          Established Tolerance (ppm)
                           Proposed Tolerance (ppm)
                          Recommended Tolerance (ppm)
Comments; Correct Commodity Definition
              Tolerances to be listed under 40 CFR §180.561(a):
Berry, low growing
subgroup 13-07G
                                      --
                                     0.15
                                     0.15


Note to Registration Division:  Tolerances have been established under 40 CFR §180.561 for residues of acibenzolar-S-methyl.  HED continues to recommend that the appropriate tolerance expression for acibenzolar-S-methyl read as follows.  

      Tolerances are established for residues of acibenzolar-S-methyl, benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester, 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 those acibenzolar-S-methyl residues convertible to benzo(1,2,3)thiadiazole-7-carboxylic acid (CGA-210007), expressed as the stoichiometric equivalent of acibenzolar-S-methyl, in or on the commodity.

      2.2.4	Revisions to Petitioned-For Tolerances

	There are no revisions to the petitioned for tolerance.

3.0	INGREDIENT PROFILE 

3.1	Chemical Identity

Table 2.	Acibenzolar-S-Methyl Nomenclature.
Chemical structure
                                       
Common name
acibenzolar-S-methyl
Company experimental name
CGA-245704
IUPAC name
benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
CAS name
S-methyl, 1,2,3-bensothiadiazole-7-carbothioate
CAS registry number
135158-54-2
End-use product (EP)
Actigard(R) 50 WG (EPA Reg. No. 100-922)
Structure of metabolite 
CGA-210007
                                       
                   1,2,3-benzothiadiazole-7-carboxylic acid


3.2	Physical/Chemical Characteristics 

      A detailed description of the physiciochemical properties of acibenzolar-S-methyl is provided in Appendix B.   Acibenzolar-S-methyl exhibits relatively low solubility in water and higher solubility in solvents.  It has a very low vapor pressure.  It does not present significant concerns for bioaccumulation based on its lipophilicity and mammalian metabolism studies.   
      
3.3		Pesticide Use Pattern

3.3.1	Registered Products	

	There are currently 7 active acibenzolar-S-methyl registrations including four Section 3 registrations and five special local needs (SLNs) or 24(c) registrations.   

Table 3.  Summary Report of Supported Registered Acibenzolar-S-methyl  Products
                                     Reg #
                                     Name
                                 Company Name
                                      %AI
100-921
Acibenzolar-S-Methyl Technical
                        Syngenta Crop Protection, Inc.
                                      99
100-922
Actigard 50WG

                                      50
100-1363
Bion 500 SF

                                      54
100-1364
Daconil Action

                                     0.11
FL030003
                        Actigard 50 WG Plant Activator

                                      50
GA020005


                                      50
NC020007


                                      50
SC030004


                                      50
VA030002


                                      50

3.3.2	Proposed New Uses 

	The Interregional Research Project No. 4 (IR-4), on behalf of the Agricultural Experiment Stations of CO, NM, UT, ID, and WA, is requesting to amend the product label for Actigard(R) (EPA Reg. No. 100-922), a water-dispersible granule (WG) formulation containing 50% acibenzolar-S-methyl to add a new use on the low growing berry crop subgroup 13-07G.  Actigard(R) is proposed for multiple foliar applications on the low growing berry subgroup using ground or aerial equipment at a maximum of 0.023 lb ai/A per application, a maximum seasonal rate of 0.188 lb ai/A, and a 0-day pre-harvest interval (PHI).  

Table 4. Proposed New Use of Acibenzolar-S-Methyl on Low Growing Berries
Applic. Timing, Type, and Equip.
                                  Formulation
                                  [EPA Reg #]
                                 Applic. Rate 
                                   (lb ai/A)
                          Max. No. Applic. per Season
                            Max. Seasonal App Rate
                                   (lb ai/A)
                                      PHI
                                    (days)
                        Use Directions and Limitations
Postemergence
Foliar or banded
Ground /Aerial
                                    50% WG
                                   [100-922]
                                  0.016-0.023
                                  8 (Implied)
                                     0.188
                                       0
Actigard(R) should be applied to healthy, actively growing plants.  Do not apply prior to thinning or within 5 days of transplanting.  Do not apply Actigard(R) to plants that are stressed due to drought, excessive moisture, cold weather, or herbicide injury, etc.  Avoid spray overlap as injury may occur.
3.4 	Anticipated Exposure Pathways

	Dietary (food and water) and residential exposures are expected based on existing uses of acibenzolar-s-methyl on low growing berries.  A residential exposure assessment is not required for this assessment because there are no residential uses or exposures associated with the proposed new use.  The short-term aggregate exposure assessment, which takes into account residential exposure plus average exposure levels to food and water, has been updated to incorporate dietary exposure from the proposed new uses on low growing berries.  

3.5	Considerations 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.  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/ASSESSMENT

4.1	Toxicology Studies Available for Analysis

      The toxicology database for acibenzolar-S-methyl is adequate for evaluating and characterizing acibenzolar-S-methyl toxicity and selecting endpoints for purposes of this risk assessment.  An immunotoxicity study required as part of new 40 CFR Part 158 data requirements for registration of a pesticide has been submitted and is being reviewed.   Based on a preliminary review, the study is acceptable and indicates no evidence of immunotoxicity.
      
4.2	Absorption, Distribution, Metabolism and Excretion

		The absorption, distribution, metabolism, and excretion of acibenzolar-S-methyl were studied in rats.  In the rat metabolism study [U-[14]C Phenyl] acibenzolar-S-methyl was administered by stomach tube to male and female rats at single doses of  0.5 (low dose) or 100 mg/kg (high dose) or at a single dose of 0.5 mg/kg following 14 repeated daily doses of 0.5 mg/kg of nonlabeled acibenzolar-S-methyl.  

		For all treatment groups, acibenzolar-S-methyl was rapidly and nearly completely absorbed (>90% of the administered dose) from the gastrointestinal tract into the general circulation.  Peak blood levels were reached at 15-30 minutes after the low dose and at 4-8 hours after the high dose.  Tissue levels of radioactivity were highest in blood, liver, kidney, skeletal muscle, heart and lungs.  Radioactivity was rapidly excreted predominantly in the urine (88-95% within 48 hours).  Only a small amount of radioactivity was excreted in the feces (3.1-4.5% within 48 hours).  Insignificant radioactivity was excreted in the expired air.  There was no significant enterohepatic circulation.  After 7 days, levels of radioactivity remaining in the tissues were extremely low.  Absorption, distribution, pharmacokinetic and excretion profiles were largely independent of dose level and sex.     

		Three urinary fractions were identified.  Fraction U3 (79-92% of the administered dose) was identified the carboxylic acid derivative of the parent.  Fraction U2 (0.4-2%) was identified as the corresponding glycine derivative of the carboxylic acid (fraction U3) derivative.   - Fraction U1 (1- 4.5%) was a mixture of at least three polar compounds that were not identified due to insufficient sample material.  No parent com - pound was detected in the urine.  Radioactivity in the feces represented <5% of the administered dose.  No single metabolite in the feces exceeded 3% of the administered dose.   Fraction F2 (0.7-3% of the administered dose) was identified as the carboxylic acid derivative of the parent.  Fraction F3 (0.1-1%) was identified as parent compound.  Frac - tion F1 (0.2-0.4%) was a very polar compound(s) that was not further identified.  The metabolic fate of acibenzolar-S-methyl was independent of dose level, sex, and pretreatment.     

		In a dermal absorption study in rats, acibenzolar-S-methyl was applied dermally to a shaved dorsal area of 10 cm² at two dose levels of 10 ug/cm² and 100ug/cm² to two groups of animals consisting of six animals/group.  Two animals from each group were sacrificed at 4, 8, and 24 hours after dosing and the amount of radioactivity in blood, urine, feces, liver, kidneys, spleen, and remaining carcass was determined.  Amount of radioactivity remaining in/on the skin and on the cover was also determined.  For the groups for which the skin was washed after 8 hours, mean dermal absorption values were 43.55% and 5.92%, and mean radioactivity remaining at the skin were 46.64%, and 90.22%, respectively at 10ug/cm² and 100 ug/cm² dose levels.  Given the uncertainty regarding actual dermal deposition under actual field conditions, it is considered appropriate to derive an estimate of dermal absorption based on the results from the low dose group (10 ug/cm[2]), since percent dermal absorption was greatest at this dose level.  Based on the likely worker exposure time frame, it is considered most appropriate to adopt the dermal absorption value calculated for the group of animals for which the skin site was washed after 8 hours.  Mean dermal absorption for this group of animals was 43.55%.  (TXR 0054866 and/or MRID 47430503)  

4.3	Toxicological Effects

	In subchronic and chronic oral studies in rats, dogs and mice, signs of mild regenerative hemolytic anemia were consistently observed in all three species.  These signs frequently included decreased erythrocyte counts, decreased hemoglobin, decreased hematocrit, increased reticulocyte counts, increased hemosiderosis in the spleen, liver and/or bone marrow, extramedullary hematopoiesis in the spleen, and increased spleen weights in both males and females.  A compensatory response (increased erythrocyte production) regularly followed the initial anemia.  Additional toxic effects observed in these same studies included decreases in body weight, body weight gain and/or food consumption.  No other significant treatment-related effects of toxicological concern were observed in these subchronic and chronic oral studies.  In a 28-day dermal study in rats, no systemic or dermal effects were observed at dose levels up to 1000 mg/kg/day, the limit dose.  No neurotoxic effects were observed at any dose in a subchronic neurotoxicity study in rats.

		Treatment-related developmental malformations, anomalies and variations were observed in a developmental toxicity study in rats at or below the no observable adverse effect level (NOAEL) for maternal toxicity.  At the highest dose tested in this study (400 mg/kg/day), both maternal toxicity (hemorrhagic perineal discharge) and considerable developmental toxicity (including total litter resorptions, fetal malformations, anomalies and variations) were observed.  The fetal malformations noted at this dose included treatment-related effects on nervous system tissues (hydrocephaly, craniorachisis and anophthalmia/ microphthalmia).  At the next lower dose tested (200 mg/kg/day), treatment-related visceral malformations and skeletal variations were demonstrated in the absence of significant maternal toxicity.  A similar increased sensitivity of fetuses or pups (as compared to adults) was not observed in a developmental toxicity study in rabbits or in 2-generation and 1-generation (range-finding) studies in rats.  In a dermal developmental toxicity study in rats, no maternal or developmental toxicity was observed at dose levels up to 500 mg/kg/day, the highest dose tested.  
      
      In a battery of mutagenicity studies, results were negative in all studies except in an in vitro chromosome aberration study in Chinese hamster ovary (CHO) cells, in which there was evidence of a clastogenic response in the absence of S-9 activation.  

		In a 2-year chronic toxicity/carcinogenicity study in rats and an 18-month carcinogenicity study in mice, acibenzolar-S-methyl was negative for carcinogenicity when administered at dose levels adequate for the testing of carcinogenic potential.   

      Acibenzolar-S-methyl showed no significant toxicity in a battery of acute toxicity tests (Toxicity Category III or IV in all tests).  Considerable skin sensitizing (contact allergenic) potential was demonstrated in a dermal sensitization study in guinea pigs.  The formulated end-use product (Actigard(TM) 50 WG) also demonstrated no significant acute toxicity; additionally, the end-use product did not show dermal sensitization in guinea pigs.

      The complete toxicity profile for acibenzolar-S-methyl is provided in Appendix A. 
      
4.4	Safety Factor for Infants and Children (FQPA Safety Factor)	

      The FQPA factor for increased susceptibility to infants and children is reduced to 1x based on the following considerations.  The toxicology database for acibenzolar-S-methyl is complete and adequate for assessing increased susceptibility under FQPA. The pre- and postnatal toxicity database for acibenzolar-S-methyl includes developmental toxicity studies in rats and rabbits, a DNT study in rats, and a two-generation reproduction toxicity study in rats.  Based on the developmental toxicity in rats and the developmental neurotoxicity studies in rats, there is concern for increased qualitative and/or quantitative susceptibility following in utero exposure to acibenzolar-S-methyl.  However, the degree of concern for the increased susceptibility seen in these studies is low as there are no residual uncertainties with regard to pre- and/or postnatal toxicity since NOAELs and LOAELs have been identified for all effects of concern, a clear dose response has been well defined, and the points of departure (PODs) selected for risk assessment are protective of the fetal/offspring effects.  Additionally, the dietary and residential risk assessments are conservative and will not underestimate dietary exposure and there are no residual uncertainties in the exposure database.  Further discussion may be found in the following sections.
      
4.4.1	Completeness of the Toxicology Database

      The toxicity database for acibenzolar-S-methyl is sufficient for a full hazard evaluation and is considered adequate to evaluate risks to infants and children.  Acceptable studies are available for developmental, reproduction, developmental and subchronic neurotoxicity.  The immunotoxicity study required under 40 CFR Part 158 data requirements has been submitted and is being reviewed.  Based on a preliminary review, the study is acceptable and indicates no evidence of immunotoxicity.
	
0.2.2 Evidence of Neurotoxicity
      
		In a developmental neurotoxicity study a maternal LOAEL was not observed.  The maternal NOAEL is 326 mg/kg/day, the highest dose tested.  No treatment-related offspring effects were seen on survival, clinical signs, FOB, developmental land marks, brain weights or neuropathology.   No conclusions can be drawn on the effects of the compound on motor activity due the lack of consistency in the observations.   Significant morphometric changes (decreased thickness of the molecular layer of the cerebellum) were observed in male offspring on PND 63 at 82 mg/kg/day.  At the high dose, treatment-related offspring effects included decreased body weights, increased auditory startle response and increased thickness in the corpus callosum in females.  In subchronic neurotoxicity study in rats no compound-related effects were observed in the FOB, motor activity, gross or neuropathology at the highest doses (575/628 mg/kg/day, M/F) tested.

0.2.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal 
      
      In the rat developmental toxicity study treatment related visceral malformations and skeletal variations were observed in fetuses at 200 mg/kg/day, the NOAEL for maternal toxicity.  In the developmental neurotoxicity study, offspring toxicity was observed at 82 mg/kg/day while no maternal toxicity was observed at 326 mg/kg/day, the highest dose tested.  Additional developmental toxicity studies in rats and rabbits and reproduction studies in rats provided no indication of increased susceptibility of rat or rabbit fetuses or neonates compared to adult animals.  Based on the results of developmental toxicity and developmental neurotoxicity studies in rats there is concern for increased qualitative and/or quantitative susceptibility following in utero exposure to acibenzolar-S-methyl.  However, the degree of concern for the increased susceptibility seen in these studies is low as there are no residual uncertainties with regard to pre- and/or postnatal toxicity since NOAELs and LOAELs have been identified for all effects of concern, a clear dose response has been well defined, and the points of departure (PODs) selected for risk assessment are protective of the fetal/offspring effects.  There are no residual uncertainties with regard to pre- and/or postnatal toxicity.  
      
      
4.4.4	Residual Uncertainty in the Exposure Database 	

		There are no residual uncertainties in the exposure database.  The dietary risk assessment is conservative and will not underestimate dietary and/or non-dietary residential exposure to acibenzolar-S-methyl.
4.5	Toxicity Endpoint and Point of Departure

	4.5.1	Dose-Response Assessment

	Toxicity endpoints and points of departure (PODs) for dietary (food and water), occupational, and residential exposure scenarios are summarized below.  A detailed description of the studies used as a basis for the selected endpoints are presented in Appendix A.  

      An acute POD of 8.2 mg/kg/day (NOAEL) was selected for all populations from an developmental neurotoxicity study in rats based on changes in brain morphometrics in the cerebellum at the LOAEL of 82 mg/kg/day.  An uncertainty factor (UF) of 100x (10x to account for interspecies extrapolation and 10x for intraspecies variation) was applied to the NOAEL to obtain an acute reference dose (aRfD) of 0.1 mg/kg/day.  Since the FQPA factor has been reduced to 1X, the acute population adjusted dose (aPAD) is equivalent to the aRfD.  The developmental neurotoxic effects of concern are presumed to occur after a single exposure and are relevant to females aged 13-50 years old since they occur in utero and to young children based on post-natal effects.  
      
[Note: In earlier risk assessments (D361893, 3/3/09), only the subpopulation females 13-50 years old was assessed for acute dietary exposure based on in utero effects.  However, based on a reevaluation of the DNT study regarding the potential for post-natal toxicity, the acute dietary analysis has been expanded to all populations in order to include children for this and more recent previous assessments (D379425, 8/19/10).]
      
      A chronic POD of 8.2 mg/kg/day (NOAEL) was selected for females 13-49 and young children years old from a developmental neurotoxicity study in rats based on changes in brain morphometrics in the cerebellum at the LOAEL of 82 mg/kg/day.  A UF 100x (10x to account for interspecies extrapolation and 10x for intraspecies variation) was applied to the dose to obtain a chronic reference dose (cRfD/cPAD) of  0.1 mg/kg/day. The effects observed in this study can occur after single or multiple doses.  Selection of a chronic endpoint from this study ensures that the chronic exposure assessment is protective of females of reproductive age and their offspring.  

[Note: In earlier risk assessments (D361893, 3/3/09), only the subpopulation females 13-50 years old was assessed for chronic dietary exposure based on in utero effects observed in the DNT study.  However, based on a reevaluation of the DNT study regarding the potential for post-natal toxicity, the chronic dietary analysis the chronic dietary exposure analysis has been revised to include an assessment of dietary risks young children using the NOAEL from the DNT study for this and more recent previous assessments (D379425, 8/19/10).]
       
	A chronic POD of 25 mg/kg/day (NOAEL) of 25 mg/kg/day was selected for adult males and females (50+ years) based on hemolytic effects seen in chronic dog study and other co-critical toxicity studies.  A UF 100x (10x to account for interspecies extrapolation and 10x for intraspecies variation) was applied to the dose to obtain a chronic reference dose (cRfD/cPAD) of 0.25 mg/kg/day.  The NOAEL of 25 mg/kg/day, taken from the chronic dog study, is based on the totality of the database with a LOAEL of 105 mg/kg/day (the lowest LOAEL is from the reproduction study) based on hemolytic anemia with compensatory response.  The NOAEL and LOAEL recommended for use in the risk assessment come from different studies conducted in different species.  Typically, a combination of NOAELs/LOAELs from studies conducted in different species is not recommended.  However, in the case of acibenzolar-S-methyl the same endpoint (hemolytic anemia) is observed across species at a relatively close range of LOAELs (105-312 mg/kg/day).  Therefore, the HED Toxicity Science Advisory Committee concluded that, in this instance, it would be appropriate to base the point of departure for the risk assessment on the NOAEL from the dog chronic toxicity study using the LOAEL from the reproduction study in rats for the basis of the endpoint (hemolytic anemia).

	Short-term incidental oral and short- and intermediate term dermal and inhalation PODs of 8.2 mg/kg/day were selected from the developmental neurotoxicity in rats. The selected endpoint is protective of offspring effects from dermal exposure. Two acceptable dermal toxicity studies, including a dermal developmental study are available.  However since neurological effects observed in the oral developmental neurotoxicity study are not measured in the dermal studies, the findings from these studies may not be protective of neurological effects.  A dermal absorption factor (DAF) is applied when dermal exposure endpoints are selected from oral toxicity studies. The dermal factor converts the oral dose to an equivalent dermal dose for the risk assessment. A dermal absorption factor of 45% was used for the dermal exposure assessment based on a dermal absorption study in rats An MOE 100 is required for incidental oral, dermal and inhalation exposure scenarios based on the conventional uncertainty factor of 100 (10x for interspecies extrapolation and 10x for intraspecies variation). 

4.5.2	Recommendations for Combining Exposure Routes

When there are potential residential exposures to the pesticide, aggregate risk assessment must consider exposures from three major sources: oral, dermal and inhalation exposures.  There is  potential residential post-application exposure to adults via the dermal route and to children via dermal and incidental oral routes of exposure.  Dietary, dermal and incidental oral endpoints for acibenzolar-S-methyl are based on common toxicological effects for females 13-49 years old and children.  Therefore, exposures from food and non-food oral and dermal routes should be aggregated for those subpopulations for this assessment.  In addition, dermal, and inhalation exposures should be aggregated for acibenzolar-S-methyl, because the same oral toxicity endpoint was selected for both exposure routes.    
	
4.5.3	Classification of Carcinogenic Potential	
       
      Acibenzolar-S-methyl has been designated by HED as "Not Likely to be Carcinogenic to Humans" in accordance with the 2005 EPA Guidelines for Carcinogenic Risk Assessment based on negative carcinogenicity studies in rats and mice and on generally negative mutagenicity studies.  
      
      
4.5.4	Summary of Points of Departure Used in Risk Assessment

      Toxicological doses/endpoints selected for the acibenzolar-S-methyl risk assessment are provided in Tables 5 and 6.
      
Table 5. Summary of Toxicological Doses and Endpoints for Acibenzolar-S-methyl  for Use in Dietary Human Health Risk Assessments
Exposure/
Scenario
                              Point of Departure
                        Uncertainty/FQPA Safety Factors
                       RfD, PAD, LOC for Risk Assessment
                        Study and Toxicological Effects
                                       
                                       
Acute Dietary (General Population)
NOAEL = 8.2 mg/kg/day
UFA= 10x
UFH=10x
FQPA SF= 1x
Acute RfD = 0.082 mg/kg/day
aPAD = 0.082 mg/kg/day
Developmental Neurotoxicity Toxicity - Rat 
Developmental LOAEL = 82 mg/kg/day based on changes in brain morphometrics in the cerebellum in offspring.  
Maternal LOAEL = was not observed 
NOAEL  = 326.2 mg/kg/day HDT
Chronic Dietary 
(Females 13-49 years & Young Children)
NOAEL = 8.2 mg/kg/day
UFA= 10x.
UFH=10x
FQPA SF= 1x
Acute RfD = 0.082 mg/kg/day
aPAD = 0.082 mg/kg/day
Developmental Neurotoxicity Toxicity - Rat 
Developmental LOAEL = 82 mg/kg/day based on changes in brain morphometrics in the cerebellum in offspring.  
Maternal LOAEL = was not observed 
NOAEL  = 326.2 mg/kg/day HDT
Chronic Dietary 
(Adult Males and Females 50+ yrs)
NOAEL = 25 mg/kg/day
UFA= 10x
UFH=10x
FQPA SF= 1x
Chronic RfD = 0.25
mg/kg/day
cPAD = 0.25 mg/kg/day
Chronic Toxicity - Dog; Co-critical; Chronic/Cancer - Rat & Mouse, Reproduction Toxicity - Rat 
LOAEL = 105 mg/kg/day based on hemolytic anemia with compensatory response.  
Incidental Oral 

NOAEL= 8.2 mg/kg/day
UFA= 10x
UFH=10x

Occupational LOC for MOE = 100
Developmental Neurotoxicity Toxicity - Rat 
Developmental LOAEL = 82 mg/kg/day based on changes in brain morphometrics in the cerebellum in offspring.  
Maternal LOAEL = was not observed 
NOAEL  = 326.2 mg/kg/day HDT
Cancer (all routes)
A "not likely" human 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).  UFL = use of a LOAEL to extrapolate a NOAEL.  UFS = use of a short-term study for long-term risk assessment.  UFDB = to account for the absence of key date (i.e., lack of a critical study).  FQPA SF = FQPA Safety Factor.  PAD = population adjusted dose (a = acute, c = chronic).  RfD = reference dose.  MOE = margin of exposure.  LOC = level of concern.  N/A = not applicable.
 


Table 6.  Summary of Toxicological Doses and Endpoints for Acibenzolar-S-methyl  for Use in Occupational Human Health Risk Assessments
Exposure/
Scenario
Point of Departure
Uncertainty Factors
LOC for Risk Assessment
Study and Toxicological Effects
Dermal Short (1-30 days) and Intermediate (1-6 months) Term
DAF = 40%
NOAEL= 8.2 mg/kg/day
UFA= 10x
UFH=10x

Occupational LOC for MOE = 100
Developmental Neurotoxicity Toxicity - Rat 
Developmental LOAEL = 82 mg/kg/day based on changes in brain morphometrics in the cerebellum in offspring.  
Maternal LOAEL = was not observed 
NOAEL  = 326.2 mg/kg/day HDT
Inhalation Short (1-30 days) and Intermediate (1-6 months) Term

NOAEL= 8.2 mg/kg/day
UFA= 10x
UFH=10x

Occupational LOC for MOE = 100
Developmental Neurotoxicity Toxicity - Rat 
Developmental LOAEL = 82 mg/kg/day based on changes in brain morphometrics in the cerebellum in offspring.  
Maternal LOAEL = was not observed 
NOAEL  = 326.2 mg/kg/day HDT
Cancer (all routes)
A "not likely" human carcinogen
DAF = Dermal Absorption Factor . 
Since no inhalation absorption data are available, toxicity by the inhalation route is considered to be equivalent to the estimated toxicity by the oral route of exposure (100% absorption factor). 

5.0	DIETARY AND DRINKING WATER EXPOSURE AND RISK ASSESSMENT

5.1 	Metabolite/Degradate Residue Profile

5.1.1	Summary of Plant Metabolism Studies

	No new plant metabolism studies were submitted.  Adequate metabolism studies with [phenyl-U-[14]C]acibenzolar-S-methyl on lettuce, tomato, rice, wheat, and tobacco have previously been submitted and reviewed.  The metabolic pathway for acibenzolar-S-methyl was similar in all tested crops and involves the hydrolysis of the S-methyl ester to form the carboxylic acid metabolite (CGA-210007).  Further hydroxylation of CGA-210007 at the C-4 and C-5 positions results in the formation of the minor metabolites CGA-324041 (all crops tested) and CGA-323060 (lettuce and tomato), which may also form acyl sugar conjugates.     

5.1.2 	Comparison of Metabolic Pathways

 	CGA 210007 represented the vast majority of the urinary radioactivity in the rat metabolism study and is therefore part of the total toxic mammalian exposure.  CGA 210007 was also the principal metabolite in plant studies.  As noted below, residues of the 4- and 5-hydroxy metabolites (CGA-324041 and CGA-323060) of CGA-210007 should be considered to be equivalent to that of the parent compound acibenzolar-S-methyl.  

5.1.3 	Environmental Fate and Transport

	Laboratory and field studies indicate that acibenzolar-S-methyl does not have a high potential to leach to ground water nor move to surface water based on its short resident times in soil and soil water.  The major degradate observed was benzo[1,2,3]thiadiazole-7-carboxylic acid (CGA 210007) in the hydrolysis and soil photolysis studies (up to 100 percent).  CGA-210007 has a longer soil residence time, lower Freundlich Kd adsorption values, and is much more water soluble than acibenzolar-S-methyl, thereby exhibiting greater potential than the parent for lateral or vertical movement after application.  Acibenzolar-S-methyl mainly degrades by photolysis and microbial action.  Aqueous and soil photolysis irradiated half lives for parent are 0.6 hours and 1 day, respectively. The aerobic soil metabolism, anaerobic aquatic metabolism and aerobic aquatic half lives are 5.3 hours, 4.0 and < 1 day(s) respectively.  Field dissipation half-lives ranged from < 1 to 27 days.  Under aerobic soil, aerobic aquatic, and anaerobic aquatic conditions acibenzolar quickly degrades to CGA 210007.  CGA 210007 is in its anionic form at environmentally significant pH's (pH 6-9) and appears to be more soluble than its parent, although measured at much lower pH's (approx. 225 ppm at 250C pH 3.6-3.8 v. 7.7 ppm at 250C pH 7.5-7.9).  CGA 210007 has longer resident field times than parent (aerobic soil half-life at 16.5 days) and based on the batch equilibrium and aged soil column leaching studies, is considerably more mobile.  Acibenzolar is not considered a bioconcentrate nor is it highly bioaccumulative.  

4..4 Residues of Concern Summary and Rationale

	The HED Metabolism Assessment Review Committee (MARC) determined that the residues of concern in plants for tolerance expression are residues convertible to benzo(1,2,3)thiadiazole-7-carboxylic acid (CGA-210007), expressed as acibenzolar-S-methyl.  For purposes of risk assessment, residues of the 4- and 5-hydroxy metabolites (CGA-324041 and CGA-323060) of CGA-210007 should also be considered.  For this purpose, the toxicity of CGA-324041 and CGA-323060 should be considered to be equivalent to that of the parent compound acibenzolar-S-methyl.  These residues occur in significant quantities in lettuce and tomato.  At this time, acibenzolar-S-methyl does not have common metabolites with other agrochemicals.  The residues of concern for drinking water are the same as those listed in the tolerance expression; that is, residues convertible to CGA-210007.  Table 7 provides a summary of the MARC decisions regarding residues of concern for acibenzolar-S-methyl.

Table 7.  Summary of MARC Decisions for Acibenzolar-S-methyl 
                                    Matrix
                            Residues of Concern [1]
                                       
                            For Risk Assessment [2]
                           For Tolerance Expression
Plants
Residues of acibenzolar-S-methyl, benzo(1,2,3) thiadiazole-7-carbothioic acid-S-methyl ester, convertible to benzo(1,2,3)thiadiazole-7-carboxylic acid (CGA-210007), expressed as acibenzolar-S-methyl &
4-hydroxy CGA-210007 (CGA-323060)
5-hydroxy CGA-210007 (CGA-324041)
Residues of acibenzolar-S-methyl, benzo(1,2,3) thiadiazole-7-carbothioic acid-S-methyl ester, convertible to benzo(1,2,3)thiadiazole-7-carboxylic acid (CGA-210007), expressed as acibenzolar-S-methyl 
[1] The residues of concern for drinking water are the same as those listed in the tolerance expression
[2] If a future petition is received for rice, residues of CGA-379019 should be included in the risk assessment

5.2	Food Residue Profile

1..1 Residues in Crops

	The registrant submitted crop field trial data from field trial conducted on strawberry as the representative crop of the low growing berry subgroup 13-07G.  Crop field trials conducted on strawberry as the representative are in accordance with the guideline for strawberry as the representative crop of the low growing berry subgroup 13-07G.  The number and locations of field trials are in accordance with OPPTS guideline requirements.  The trials reflect the proposed maximum seasonal rate and pre-harvest interval (PHI), and there are adequate storage stability data to validate sample storage conditions and duration.  The majority of the tank mixes contained an adjuvant.  The maximum residues of acibenzolar-S-methyl (i.e., residues convertible to CGA-210007) were 0.087 ppm in/on strawberry.  The residue data were entered into the OECD MRL Calculator.  The available strawberry field trial data are deemed adequate to support a tolerance of 0.15 ppm for residues of acibenzolar-S-methyl in/on the low growing berry subgroup 13-07G.

	Adequate confined rotational crop data have been submitted previously.  Based on the available field rotational crop data for wheat, turnip, and lettuce, tolerances are not needed for rotational crops provided a 30-day plant-back interval is established for all crops not listed as target crops.  The existing rotational crop restrictions on the Actigard(R) label are adequate.

5.3 	Water Residue Profile
    
5.3.1	Estimated Drinking Water Concentrations 

      Drinking water residues provided by the Environmental Fate and Effects Division (EFED) (D353745, R. Miller, 9/2/10) were incorporated directly into the acute and chronic dietary analyses.  The surface water estimates were based on EFED's Tier II model Pesticide Root Zone Model.  The residues of concern for drinking water include acibenzolar-S-methyl and residues convertible to CGA-210007.  The estimated drinking water concentrations (EDWCs) for use of acibenzolar-S-methyl on cucumbers were used for the drinking water assessment because this use represents the highest use rate of the existing and proposed registered uses.  The SCI-GROW estimated ground water drinking water concentrations for acibenzolar is not expected to exceed 1.25 x 10[-5] ug/L and CGA 210007 is not expected to exceed 8.12 x 10[-2] ug/L.  Surface water EDWCs for acibenzolar-S-methyl, which were used in the exposure assessment, are provided in Table 8.  CGA-210007 drinking water residues were included in the dietary exposure assessment as acibenzolar-S-methyl equivalents. 

 Table 8.  EDWCs for use in screening the human health risk assessment (FL Cucumber)
Drinking Water Source
 Chemical
 1-in-10 year annual acute conc. (μg/L)
 1-in-10 year avg annual (chronic) concentration (μg/L)
 30-year avg. annual concentration(μg/L)
Surface Water
Acibenzolar-S-Methyl[1]
                                     0.72
                                     0.02
                                     0.02
Surface Water
CGA 210007[2]
                                     20.02
                                     8.09
                                     4.68
[1] Aerial application method
[2] Degradation product formed after application 

5.4 	Dietary and Drinking Water Exposure and Risk

	Refined acute and screening-level chronic dietary and drinking water exposure and risk assessments were conducted using the Dietary Exposure Evaluation Model with the Food Commodity Intake Database (DEEM-FCID(TM)).  Dietary risk assessment incorporates both exposure and toxicity of a given pesticide.  For acute and chronic dietary assessments, the risk is expressed as a percentage of a maximum acceptable dose (i.e., the dose which HED has concluded will result in no unreasonable adverse health effects).  This dose is referred to as the population adjusted dose (PAD).  The PAD is equivalent to the reference dose (RfD) divided by the additional Safety Factor, if applied. For acute and non-cancer chronic exposures, HED is concerned when estimated dietary risk exceeds 100% of the PAD.  

	Available lettuce metabolism data for a 7-day PHI were used to estimate the relative abundance of the hydroxy metabolites (CGA-323060 and CGA-324041) to the residues of acibenzolar-S-methyl convertible to the benzo(1,2,3) thiadiazole-7-carboxylic acid (CGA-210007).  Based on the lettuce metabolism data, a factor of 1.5X was applied to estimates of acibenzolar-S-methyl residues to account for all of the residues of concern for dietary risk (including CGA-323060 and CGA-324041).  

5.4.1	Acute Dietary and Drinking Water Analysis

      A refined (probabilistic) acute dietary exposure analysis was performed for the general population and all population subgroups.  The acute analysis assumed a distribution of residues based on field trial data.  Empirical and DEEM default processing factors were used to modify the field trial data.  Maximum screening-level percent crop treated estimates were used for commodities for which data were available.  If no percent crop treated data were available, 100% crop treated was assumed.  The acute analysis incorporated the entire time distribution of EDWCs adjusted to account for CGA-210007.  Acute dietary risk estimates are not of concern for general population or other population subgroups.  The subgroup with the highest risk estimate was children 3-5 years old with a 99.9[th] percentile acute exposure estimates of 35% of the aPAD.  The 99.9[th] percentile aPAD for the general U.S. population was 17%. 
      
Table 9:  Summary of Acute Dietary Exposure and Risk for Acibenzolar-S-Methyl
Population Subgroup
                               aPAD (mg/kg/day)
                           Acute (99[th] Percentile)

                                       
                             Exposure (mg/kg/day)
                                    % aPAD
General U.S. Population
                                     0.082
                                   0.014091
                                      17
All Infants (< 1 year old)
                                       
                                   0.012951
                                      16
Children 1-2 years old
                                       
                                   0.025042
                                      31
Children 3-5 years old
                                       
                                   0.028536
                                      35
Children 6-12 years old
                                       
                                   0.019902
                                      24
Youth 13-19 years old
                                       
                                   0.015095
                                      18
Adults 20-49 years old
                                       
                                   0.009060
                                      11
Adults 50+ years old
                                       
                                   0.010614
                                      13
Females 13-49 years old
                                       
                                   0.009540
                                      12


5.4.2	Chronic Dietary and Drinking Water Analysis

       A conservative chronic dietary exposure analysis was performed for the general U.S. population and various population subgroups.  Tolerance level residues and 100 crop treated assumptions were used.  DEEM default and empirical processing factors were used to modify the tolerance values.  The chronic analysis incorporated the 10 year average surface drinking water estimate from application of acibenzolar-S-methyl to cucurbit vegetables.  The population subgroups females 13-49 and young children had a more protective chronic population adjusted dose (cPAD, 0.082 mg/kg/day) than the general U.S. population and all other population subgroups (0.25 mg/kg/day).  Chronic dietary risk estimates are not of concern for general population or other population subgroups.  The subgroups with the highest risk estimate were children 1-2 and 3-5 years old with a cPAD of 11%.  The cPAD for the general U.S. population was 2%. 
      
Table 10:  Summary of Chronic Dietary Exposure and Risk to Female and Child Population for Acibenzolar-S-Methyl 
Population Subgroup
                               cPAD (mg/kg/day)
                                    Chronic

                                       
                             Exposure (mg/kg/day)
                                    % cPAD
Females 13-49 years old
                                     0.082
                                   0.003646
                                       4
All Infants (< 1 year old)
                                       
                                   0.003808
                                       5
Children 1-2 years old
                                       
                                   0.009207
                                      11
Children 3-5 years old
                                       
                                   0.008706
                                      11
Children 6-12 years old
                                       
                                   0.008706
                                       7
      
Table 12:  Summary of Chronic Dietary Exposure and Risk to Adults 50+ and Adult Males (13+) Acibenzolar-S-Methyl
                              Population Subgroup
                               cPAD (mg/kg/day)
                                    Chronic
                                       
                                       
                             Exposure (mg/kg/day)
                                    % cPAD
General U.S. Population
                                     0.25
                                   0.004366
                                       5
Males 13-19
                                       
                                   0.003893
                                       2
Males 20+
                                       
                                   0.003654
                                       2
Adults 50+ years old
                                       
                                   0.003852
                                       2
      
6.0 	RESIDENTIAL EXPOSURE AND RISK ASSESSMENT

      A new residential exposure assessment is not required for the proposed new use of acibenzolar-S-methyl on low growing berries because there are no homeowner uses and/or applications to residential areas associated with this use.
   
6.1	Residential Bystander Postapplication Inhalation Exposure

      Based on the Agency's current practices, a quantitative residential bystander postapplication inhalation exposure assessment was not performed for acibenzolar-S-methyl at this time.  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 acibenzolar-S-methyl.

6.2	Spray Drift

      Spray drift is always a potential source of exposure to residents nearby to 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 acibenzolar-S-methyl.  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.
      
      Although a quantitative residential post-application inhalation exposure assessment was not performed as a result of pesticide drift from neighboring treated agricultural fields, an inhalation exposure assessment was performed for flaggers.  This exposure scenario is representative of a worse case inhalation (drift) exposure and may be considered protective of most outdoor agricultural and commercial post-application inhalation exposure scenarios. 
 
 7.0 	AGGREGATE EXPOSURE AND RISK ASSESSMENT 
 
      	In accordance with the FQPA, when there are potential residential exposures to a pesticide, aggregate risk assessment must consider exposures from three major routes: oral, dermal, and inhalation.  There are three sources for these types of exposures:  food, drinking water, and residential uses.  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.  Acute and chronic aggregate exposures include food plus drinking water exposures.    Acute and chronic aggregate risks are not of concern.


7.1	Short- Term Aggregate Risk

      To assess short--term aggregate risk likely to result from the new and existing uses, HED combined average food and water exposure values with estimates of residential exposure for adult handlers and with residential exposure for adult and child post-application activities Tables 11 and 12.  Exposure for adult and child golfers was used to aggregate adult post-application dermal exposure with dietary and drinking water exposure.  The aggregate exposure assessment for children combines dermal and incidental oral post-application exposure with food and water exposure.  
      
Table 11.  Short-Term Aggregate Risk Calculations  - Residential Handler LOC =100
                              Population Subgroup
                                    Average
                           Food & Water Exposure
                                   mg/kg/day
                                  Residential
                                  Exposure[1]
                                   mg/kg/day
                                 Aggregate MOE
                                   (food and
                                residential)[2]
Females 13-49 yrs
                                    0.0038
                                    0.0073
                                      700
[1] Residential Exposure = [Dermal exposure + Inhalation Exposure].  
[2] Aggregate MOE = [NOAEL (8.2 mg/kg/day (Avg Food & Water Exposure + Residential Handler Exposure)]

Table 12.  Short-Term Aggregate Risk Calculations  - Residential Post-Application Exposure LOC =100
                              Population Subgroup
                                    Average
                           Food & Water Exposure
                                   mg/kg/day
                            Residential Exposure[1]
                                   mg/kg/day
                                 Aggregate MOE
                                   (food and
                                residential)[2]
Females 13-49 yrs
                                    0.0038
                                    0.00135
                                     1600
Children  6-12 yrs
                                    0.0059
                                    0.00194
                                     1000
Children 1-2 yrs
                                    0.0093
                                    0.0009
                                      800
Children 3-5 yrs
                                    0.0088
                                       
                                      850
[1] Residential Exposure = [Dermal exposure + Inhalation Exposure].  
[2] Aggregate MOE = [NOAEL (8.2 mg/kg/day (Avg Food & Water Exposure + Residential Exposure)]  Adult residential post-application exposure from golfer scenario.  Child residential exposure includes incidental oral and dermal exposures.

8.0	CUMULATIVE RISK

      Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the Agency consider "available information" concerning the cumulative effects of a particular pesticide's residues and "other substances that have a common mechanism of toxicity."

	EPA does not have, at this time, available data to determine whether acibenzolar-S-methyl has a common mechanism of toxicity with other substances.  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 acibenzolar-S-methyl and any other substances and, acibenzolar-S-methyl does not appear to produce a toxic metabolite produced by other substances which have tolerances in the U. S.  For the purposes of this tolerance reassessment action, therefore, EPA has not assumed that acibenzolar-S-methyl 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 OPP concerning common mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA's website at http://www.epa.gov/fedrgstr/EPA_PEST/2002/January/Day_16/.

9.0	OCCUPATIONAL EXPOSURE/RISK CHARACTERIZATION

9.1	 Exposure Scenarios

		Occupational handler and post-application exposure scenarios were assessed for the risk assessment of the proposed new uses on low growing berries.  Based on the product labels and information provided by the registrant, short- and intermediate-term exposure is assessed for occupational handlers and post-application activities.  Dermal and inhalation exposures to workers are aggregated for acibenzolar-S-methyl because the toxicity endpoints for these exposure routes are based on common toxicological effects.

9.2	Handler Exposure 

		The term "handler" applies to individuals who mix, load, and apply the pesticide product. The following handler exposure scenarios were assessed for proposed new acibenzolar-S-methyl uses.

9.2.1 	Handler Exposure Scenarios

	-	Open mixing/loading (dry flowable) for aerial application;
	-	Open mixing/loading (dry flowable) for groundboom application;
	-	Applying sprays via aerial applications;
	-	Applying sprays via groundboom applications
	-	Flagging for aerial applications
9.2.2	Handler Exposure Data

      No chemical-specific handler exposure data were submitted in support of this registration.  It is the policy of the HED to use surrogate data from the US Environmental Protection Agency, Office of Pesticide Programs , Occupational Pesticide Handler Unit Exposure Surrogate Reference Table, June 21, 2011, which summarizes and extracts surrogate data 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. Some of these data, such as the industry task force data, are compensatory, subject to the data protection provisions of FIFRA.  HED policies on use of surrogate data, including their sources, are presented on the internet at http://www.epa.gov/pesticides/science/handler-exposure-data.html . The "Occupational Pesticide Handler Unit Exposure Surrogate Reference Table" may be found at http://www.epa.gov/pesticides/science/handler-exposure-table.pdf.

9.2.3 	Handler Exposure Assumptions

		The following standard exposure assumptions were used in estimating risks to workers from exposure to acibenzolar-S-methyl for the proposed new use on low growing berries. The daily areas treated were defined for each handler scenario by determining the amount that can be reasonable treated in a single day.  When possible, the assumptions for daily areas treated are taken from HED's ExpoSAC Policy #9 (: "Standard Values for Daily Acres Treated in Agriculture", which was revised on July 5, 2000.

		::	Average body weight of an adult handler is 60 kg.
		::	Exposure duration is short-term and intermediate-term for all workers assessed.  
 		::	Maximum application rates as determined by label review were used for all types and methods of application. The maximum single application rate for low growing berries is 0.023 lbs ai per acre.
		::	Estimated dermal absorption is 44%
		::	SOP daily volumes handled and/or area treated used for the scenarios assessed are: 
			- 	80 acres treated per day for groundboom mixer/loader and applicator
			-  	350 acres treated per day for applications with fixed wing aircraft and flagging 

9.2.4 	Handler Exposure and Risk Estimates
 			
 		A target LOC or MOE of 100 is considered adequate for inhalation and dermal exposure. All worker exposures are assessed as short-and intermediate-term based on label prescribed uses and expected exposure durations.  Dermal and inhalation exposures to workers should be aggregated for acibenzolar-S-methyl because the same oral toxicity endpoint was selected for these exposure routes.  Exposure and risk estimates indicate MOEs are not of concern (MOEs > 100) at the maximum use rate for occupational handler activities for the proposed new uses.  A summary of occupational handler exposure and risk calculations, assumptions, and results is provided in Table 13. 

Table 13: Short/Intermediate-Term  Occupational Handler Exposure and Risk for Acibenzolar-S-Methyl for Application to Low Growing Berries
Exposure Scenario [1]
App Rate 
lbai/A[2]
Acres Treatd /day
                          Unit Exposure[3] (ug/lb ai)
                             Dose (mg/kg/Day)[4,5]
                                    MOEs[6]



Dermal 
Inhal 
Dermal
Inhal
Dermal
Inhal
Combined Derm & Inhal



Baseline
Gloves

BL
G

BL
G

BL
G
                          Mixer/Loader (dry flowable)
Aerial 
                                     0.023
                                      350
                                      227
                                      52
8.96
0.0133
0.0030
0.0012
                                      620
                                     2700
                                     6800
                                      570
                                     1800
Ground
boom

                                      80



0.0030
0.0006
0.0003
                                     2500
11000
27000
                                     2500
8000
                           Applicator (dry flowable)
Aerial
                                     0.023
                                      350
                                     0.005
                                    Eng. C
                                      N/A
0.068
0.0003
NA
9E-06
28000
NA
900000
25000
NA
Ground
boom
                                       
                                      80
                                     78.6
                                     16.1
0.0003
0.0011
0.0002
1E-05
                                     7800
35000
790000
                                     7700
37000
                            Flagger (dry flowable)
Aerial 
                                     0.023
                                      350
                                      11
                                      12
                                     0.35
                                    0.0006
                                    0.0007
                                     5E-05
                                     13000
                                     12000
                                    170000
                                     12000
                                     11000
[1]  Use patterns are from the proposed labels 
[2] Application rates are based on maximum values based on proposed label. 
[3] Unit exposures for baseline (no personal protective equipment (PPE) and PPE (e.g., gloves).  Values are reported in the PHED Surrogate Exposure Guide dated August 1998 or are from data submitted by the Outdoor Residential Exposure Task Force dated May 2000.  
4 Inhalation dose (mg/kg/day) = [unit exposure (ug/lb ai) * 0.001 mg/ug unit conversion * Inhalation absorption (100%) * Application rate (lb ai/acre or lb ai/gallon) * Daily area treated/amount handled (acres or gallons)] / Body weight (60 kg).
[5] Dermal Dose (mg/kg/day) = [unit exposure (mg/lb ai) * Application rate (lb ai/acre or lb ai/gallon) * Daily area treated/amount handled (acres or gallons) * Dermal Absorption Factor (44%)] / Body weight (60 kg).
[6]  Aggregate MOE = NOAEL (8.2 mkd)/Daily Inhalation Dose + Daily Dermal Dose

9.3	Post Application Exposure 
      
      HED uses the term "post-application" to describe those individuals who can be exposed to pesticides after entering areas previously treated with pesticides and performing certain tasks or activities.  For acibenzolar-S-methyl, post-application dermal exposure to field workers following foliar applications is probable since acibenzolar-S-methyl is a fungicide that can be applied to all stages of growth.  Since no post-application data were submitted in support of the registration action, dermal exposures during post-application activities were estimated using dermal transfer coefficients from the Science Advisory Council for Exposure Policy Number 3 (http://www.epa.gov/pesticides/science/exposac_policy3.pdf).  
      
      Based on the Agency's current practices, a quantitative occupational post-application inhalation exposure assessment was not performed for acibenzolar-S-methyl at this time.  However, there are multiple potential sources of post-application inhalation exposure to individuals performing post-application activities in previously treated fields.  These potential sources include volatilization of pesticides and re-suspension of dusts and/or particulates that contain pesticides.  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 as well as available postapplication inhalation exposure data generated by the Agricultural Reentry Task Force and may, as appropriate, develop policies and procedures, to identify the need for and, subsequently, the way to incorporate occupational post-application 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 occupational post-application inhalation exposure assessment for acibenzolar-s-methyl.
      
9.3.1	Post Application Exposure Scenarios
 
		::	Handweeding
		::	Scouting 
		::	Transplanting
		::	Hand harvesting, scouting, and raking
		::	Irrigating
	
9.3.2	Post Application Exposure Assumptions

		::	Average body weight is 60 kg.
		::	Transferrable residue is application rate times the fraction initially available (20%). 
		::	Maximum transfer coefficient for post-application activities is 1900 cm[2]/hour (irrigation).
		::	Exposure duration is 8 hours day. 
		::	Exposure is assumed to occur on the day of application (day 0). 

9.3.3 	Post-Application Exposure and Risk Estimates
 
 		A target LOC or MOE of 100 is considered adequate for dermal exposure.  Exposure and risk estimates indicate MOEs are not of concern (MOEs > 100) at the maximum use rate for occupational post-application exposure activities for the proposed new uses.  A summary of post-application exposure and risk calculations, assumptions, and results is provided in Table 14.


Table 14.  Summary of Estimated Post-application MOEs for Agricultural Crops
                                     Crop
                               Application Rate
                                  (lb ai/A) 1
                                    DAT[2]
                                     DFR 3
                                  (μg/cm[2])
                                     TC 4
                                  (cm[2]/hr)
                                  Activity 4
                                  Short-/Int-
                                  Term MOE 5
Low Growing Berries
                                     0.023
                                       0
                                     0.052
                                      70
                                 Hand weeding
                                    39,000

                                       
                                       
                                       
                                      210
                                   Scouting
                                    13,000

                                       
                                       
                                       
                                      230
                                 Transplanting
                                    12,000

                                       
                                       
                                       
                                     1,100
                       Hand harvesting, Scouting, Raking
                                     2,500

                                       
                                       
                                       
                                     1,900
                                  Irrigation
                                     1,400
[1] Maximum application rate from proposed label   
    2 DAT = Days after treatment needed to reach the LOC of 100; DAT 0 = the day of treatment/ assumed to be approx. 12 hours.  
    3 DFR (ug/cm[2]) = dislodgeable foliar residues corresponding to DAT, based on 20% of application rate.
    4 TC (cm[2]/hr) = transfer coefficients and associated activities from ExpoSAC Policy Memo #003.1, 8/17/2000.
   5 MOE = MOE on the corresponding DAT.  MOE = NOAEL / Daily Dose.  
    Daily Dose = [(DFR x TC x 40% Dermal absorption x 8-hr Exposure Time)] / [(CF: 1000 ug/mg) x (60-kg Body Weight)   
  Short-/intermediate-term NOAEL = 8.2 mg/kg/day.  The LOC is 100

9.3.4 	Restricted Entry Interval

	Acibenzolar-S-methyl has been classified in Toxicity Category III for acute dermal and eye irritation and Category IV for primary skin irritation.  Per the Worker Protection Standard (WPS), a 12-hr restricted entry interval (REI) is required for chemicals classified under Toxicity Category III/IV.  

10.	REFERENCES

Acibenzolar-S-Methyl.  Petition for the Establishment of Permanent Tolerances on Low Growing Berries (Subgroup 13-07G).  Summary of Analytical Chemistry and Residue Data, T. Morton, D386364, 2/23/12

Acibenzolar-S-Methyl  - Acute and Chronic Dietary and Drinking Water Exposure and Risk Assessment to support a Section 3 Request for Use of Acibenzolar-S-Methyl on Low Growing Berries Crop Subgroup 13-07G, T. Morton, D387069, 2/23/12

Occupational Exposure Assessment to Support the Request for Registration of Acibenzolar-S-Methyl on Low Growing Berries (subgroup 13-07 G), J.S. Miller, D387841, 2/23/12 

Acibenzolar: Subchronic Neurotoxicity Study in Rats, K. Oo, D392694, 8/3/11 

Absorption and Excretion of [U-[14C]]Phenyl CGA 245704 in the  Rat after Dermal Application, K. Oo, D392695, 8/3/11.

Acibenzolar, Drinking Water Exposure Assessment  -  R. Miller, D353745, 9/2/10

APPENDICES

   A. TOXICOLOGY DATA SUMMARY

A.1 		Guideline Data Requirements

                                 Guideline No.
                                                 Study Type
                                   Technical
                                   MRID No.


                                   Required
                                   Submitted
                                       
                  870.3100
                  
                  870.3150
                  870.3200
                  
                  870.3250
                  870.3465
                  Subchronic (Oral) Toxicity - Rodent	
                  
                  Subchronic (Oral) Toxicity - Non-Rodent	
                  21/28-Day Dermal Toxicity	
                  
                  90-Day Dermal Toxicity		
                  90-Day Inhalation Toxicity 	
                                       Y
                                       
                                       Y
                                       N
                                       
                                       N
                                       N
                                       Y
                                       
                                       Y
                                       Y
                                       
                                       N
                                       N
                                   44014230 
                                   44014228
                                   44014232
                                   42090013
                                   46950310
870.3700a

870.3700b
870.3800
                  Prenatal Developmental Toxicity - Rodent	
                  
                  Prenatal Developmental Toxicity - Non-Rodent	
                  Reproduction and Fertility Effects	
                                       Y

                                       Y
                                       Y
                                       Y
                                       
                                       Y
                                       Y
                                   44014236
                                   45089701
                                   44014237
44014241
870.4100a
870.4100b
870.4200a
870.4200b
870.4300
                  Chronic (Oral) Toxicity - Rodent	
                  Chronic (Oral) Toxicity - Non-Rodent (Dog)	
                  Carcinogenicity - Rat..................		
                  Carcinogenicity - Mouse	
                  Combined Chronic Toxicity /Carcinogenicity
                                       Y
                                       Y
                                       Y
                                       Y
                                       Y
                                       Y
                                       Y
                                       Y
                                       Y
                                       Y
                                   44014325
                                   44014234
                                   44014243
                                   44014325
                                   44014325
870.6100a
870.6100b
870.6200a
870.6200b
870.6300
870.7800
                  Neurotoxicity - Acute Delayed Neurotox.- Hen	
                  Neurotoxicity  - Subchronic - Hen	
                  Neurotoxicity - Acute - Rat	
                  Neurotoxicity -Subchronic - Rat	
                  Developmental Neurotoxicity..................
                  Immunotoxicity.............................	
                                       N
                                       N
                                       Y
                                       Y
                                       N
                                       Y
                                       N
                                       N
                                       Y
                                       Y
                                       Y
                                      Y*
                                      ---
                                      ---
                                      ---
                                   45713601
                                   46046401
                                   48688301
   oo  Studies have been submitted and are under review 


A.2		Toxicity Profiles 

Table 2  Acute Toxicity Profile Acibenzolar-S-methyl  
                             Acibenzolar-S-methyl
                                Guideline No./
                                  Study Type
                                   MRID No.
                                    Results
                               Toxicity Category
870.1100 Acute oral toxicity
                                   44014214
LD50 >5000 mg/kg
                                      IV
870.1200 Acute dermal toxicity
                                   44014216
LD50 >2000 mg/kg
                                      IV
870.1300 Acute inhalation toxicity
                                   44014219
LC50 >5.022 mg/L
                                      IV
870.2400 Acute eye irritation
                                   44014220
minimal
                                      III
870.2500 Acute dermal irritation
                                   44014220
slight
                                      IV
870.2600 Skin sensitization
                                   44014225
positive
                                      NA
                                 Actigard 50WG
870.1100 Acute oral toxicity
                                   44014215
LD50 >5000 mg/kg
                                      IV
870.1200 Acute dermal toxicity
                                   44014217
LD50 >2000 mg/kg
                                      IV
870.1300 Acute inhalation toxicity
                                   44014218
LC50 >2.79 mg/L
                                      IV
870.2400 Acute eye irritation
                                   44014221
minimal
                                      III
870.2500 Acute dermal irritation
                                   44014223
moderate
                                      III
870.2600 Skin sensitization
                                   44014224
negative
                                      NA

Table 3  Subchronic, Chronic and Other Toxicity Profile for Acibenzolar-S-methyl 
                                 Guideline No.
                                  Study Type
                                MRID No. (year)
                                Classification
                                  Dose Levels
                                    Results
870.3100
90-day oral toxicity rodents (rat)
44014230 (1993)
Acceptable/Guideline
M : 0, 2.42, 24.6, 126, 516 mg/kg/day;
F: 0, 2.64, 26.3, 131, 554 mg/kg/day
NOAEL:126 mg/kg/day Males or M); 131 mg/kg/day (Females or F)
LOAEL:516 mg/kg/day (M); 554 mg/kg/day females based decreased body weight, reduced food intake and efficiency and increased liver and spleen weights
870.3150
90-day oral toxicity non-rodents (dog)
44014232 (1994)
Acceptable/Guideline
M & F: 0, 10, 50, 200 mg/kg/day (capsules)

NOAEL:50 mg/kg/day (M & F)
LOAEL:200 mg/kg/day (F) based on regenerative hemolytic anemia evidenced by decreased erythroid parameters, pigmentation in the liver and spleen, splenic congestion, and by activation of medullary (bone marrow) and extramedullary (splenic) erythropoiesis.
870.3100
90-day oral toxicity rodents (mice)
44014228 (1993)
Acceptable/Guideline
0, 30.6, 152, and 624 mg/kg/day for males, and 0, 47.4, 220, and 803 mg/kg/day for females, respectively.
NOAEL: 30.6 mg/kg/day (M); 47.4 mg/kg/day (F)
LOAEL:152 mg/kg/day (M); 220 mg/kg/day females based decreased body weight and body weight gain, increased spleen weight and microscopic changes in the spleen
870.3200
21/28-day dermal toxicity (rat)
44014233 (1994)
Acceptable/guideline
M & F: 0, 10, 100, 1000 mg/kg/day
NOAEL = 1000 mg/kg/day
LOAEL = not identified

870.3700a
Developmental Toxicity in rodents (rat)
44014236 (1994)
Acceptable/guideline
F: 0,10, 50, 200, 400 mg/kg/day

Maternal NOAEL 200 mg/kg/day
Developmental NOAEL 50 mg/kg/day
Maternal LOAEL 400 mg/kg/day based on clinical signs of hemorrhagic perineal discharge
Developmental LOAEL 200 mg/kg/day based on developmental malformations, anomalies, and skeletal variations
870.3700a
Developmental Toxicity in rodents (rat)
45089701 (1998)
Acceptable/guideline
of 0, 10, 75, 150, or 350 mg/kg/day
Maternal NOAEL 350 mg/kg/day
Developmental NOAEL 200 mg/kg/day
Maternal LOAEL  -  not established (>350) 
Developmental LOAEL 350 mg/kg/day based on increased incidences of rudimentary and long lumbar ribs
870.3700b
Developmental Toxicity in non- rodents (rabbit)
44014237 (1994)
Acceptable/guideline
F: 0, 10, 50, 300, 600 mg/kg/day

Maternal NOAEL = 50 mg/kg/day
LOAEL = 300 mg/kg/day based on mortality, clinical signs of toxicity, decreased maternal body weight and food consumption.
Developmental NOAEL = 300 mg/kg/day
LOAEL = 600 mg/kg/day based on a marginal increase in vertebral anomalies.
870.6300
Developmental Neurotoxicity Toxicity in rodents (rat)
46046401 (2002)
Acceptable/non-guideline
0/0, 8.2/15.5, 82.0/153.6, and 326.2/607.8 mg/kg/day [gestation/lactation].  
Maternal NOAEL 326.2 mg/kg/day
Developmental NOAEL 8.2 mg/kg/day
Maternal LOAEL Not Observed
Developmental LOAEL 82 based on changes in brain morphometrics in the cerebellum
870.3800
Reproduction and fertility effects in rats
4014241 (1995)
Acceptable/guideline
M & F: 0, 1-3, 11-31, 105- 288, 223-604  mg/kg/day

Parental/Systemic NOAEL = 11-31 mg/kg/day
LOAEL = 105-288 mg/kg/day based on increased weights and hemosiderosis of the spleen.
Reproductive NOAEL = 223-604 mg/kg/day
LOAEL > 223-604 mg/kg/day based on no effects.
Offspring NOAEL = 11-31 mg/kg/day
LOAEL = 105-288 mg/kg/day based on reduced pup body weight gains and lower pup body weights during lactation.
870.6200b 
Subchronic Neurotoxicity
45713601 (1997)
acceptable/nonguideline pending submission of positive control data for neuropathological and FOB assessments
0/0, 24/26, 126/143, and 575/628 mg/kg/day [M/F], respectively
The study may be upgraded pending submission of acceptable positive control data.
NOAEL 126/143 mg/kg/day, M/F
LOAEL 575/628 mg/kg/day, M/F based on decreased body weights, body weight gains, and food consumption.  
870.4100
Chronic toxicity in dogs
44014234 (1996)
Acceptable/guideline
M & F: 0, 5, 25, 200 mg/kg/day (capsules)
NOAEL 25 mg/kg/day
LOAEL 200 mg/kg/day based on hematological effects, hemosiderosis of the liver and spleen, extraniedullary hematopoiesis in the spleen, and increases in liver weight.
870.4300
Chronic toxicity/ carcinogenicity in rodents (mice)
44014325 (1996)
Acceptable/guideline
M: 0, 1.14, 11.1, 237, 698 mg/kg/day
F: 0, 1.14, 10.8, 234, 696 mg/kg/day
NOAEL 11.1/10.8 mg/kg/day, M/F
LOAEL 237/234 mg/kg/day based on mild hemolytic anemia and related microscopic changes (hemosiderosis) in the spleen, liver, and bone marrow and extramedullary hematopoiesis in the spleen.
870.4200
Carcinogenicity study in rat
44014243 (1996)
Acceptable/guideline
M: 0, 0.77, 7.77, 96.9, 312 mg/kg/day
F: 0, 0.90, 9.08, 111, 388 mg/kg/day
NOAEL 96.9/111 (M/F)
LOAEL 312/388 / 191.0 mg/kg/d (M/F) based on decreased body weights, body weight gain and food efficiency, mild hemolytic anemia, and increased incidence of alveolar foam cells (females only).
870.5100
Bacterial reverse mutation assay (Ames test)
441014247 (1993)
Acceptable/guideline
312.5, 625, 1250, 2500, 5000 g/plate
Negative with and without S-9 activation at 5000 g/plate and less.
870.5100
Bacterial reverse mutation assay (Ames test)
Test Material: CGA- 362020 (isomer of acibenzolar-S-methyl)
44537025 (1998)
Acceptable/guideline
61.73 to 5000 g/plate (+S-9)
30-86 to 2500 g/plate (-S-9)
Positive in S. typhimurium strain TA1537 at 277.8 g/plate and higher in the absence of S-9.  Negative with S-9 activation at 5000 g/plate and less.
870.5100
Bacterial reverse mutation assay (Ames test)
Test Material: NOA- 419191 (by-product of acibenzolar-S-methyl)
44537024 (1998)
Acceptable/guideline
312.5 to 5000 g/plate (+- S-9)
Negative with or without S-9 activation at 5000 g/plate and less
870.5100
Bacterial reverse mutation assay (Ames test)
Test Material: CGA- 323060 (plant metabolite of acibenzolar-S-methyl)
44537026 (1997)
Acceptable/guideline
312.5 to 5000 g/plate (+- S-9)
Negative with or without S-9 activation at 5000 g/plate and less
870.5300
In vitro mammalian gene mutation assay 
44014246 (1993)
Acceptable/guideline
3.70 to 100 g/ml (-S-9), 37.04 to 1000 g/ml (+S-9)
Negative with S-9 activation up to 1000g/ml.   Negative without S-9 activation up to 100g/ml.
Compound tested to cytotoxic concentrations.
870.5375
In vitro mammalian chromosome aberration (CHO cells)
44014245 (1993)
Acceptable/guideline
7.5 to 60 g/ml (-S-9 and +S- 9)
Suggestive of clastogenicity in the absence of S-9 activation at 30 and 60 g/mL at the 18-hour cell harvest time; effect observed only in the presence of cytotoxicity.  Increase in polyploid cells at 30 and 60 g/mL at the 42 hour harvest time both with and without S-9.  Evidence of cell cycle arresting activity at G2.
870.5395
Mammalian erythrocyte micronucleus test
44014244 (1993)
Acceptable/guideline
1000, 2000, 4000 mg/kg (oral gavage)
Negative at 16, 24, and 48, hour sacrifices.
870.5550
UDS in primary rat hepatocytes
44014248 (1993)
Acceptable/guideline
9.77 to 500 g/ml
Negative at 500 g/ml and less.
870.7485
Metabolism and pharmacokinetics
rats
44014250 (1995)
Acceptable/guideline
0.5, 100 mg/kg
Following oral treatment of rats, acibenzolar-S-methyl was rapidly and nearly completely (>90% of administered dose) absorbed from the gastrointestinal tract into the general circulation.   The majority  (88- 95%) of the administered dose was excreted in the urine within the first 48 hours.  The major metabolite (79- 92%) in the urine was the carboxylic acid derivative of the parent.
870.7600
Dermal penetration
NA
NA
Special studies:

28-Day dietary 
rats



44014227 (1992)
Acceptable/nonguideline
M: 0, 45.9, 403, 1070 mg/kg/day; F: 0, 44.8, 376, 1000 mg/kg/day


NOAEL =  M: 403 mg/kg/day; F: 376 mg/kg/day
LOAEL = M: 1070 mg/kg/day; F: 1000 mg/kg/day based on decreased mean body weights, decreased liver weights, altered hematology parameters accompanied by increased spleen weights.
28-Day oral gavage
rats
44014229 (1993) 
Acceptable/nonguideline
0, 10, 100, 800 mg/kg/day 
NOAEL = 100 mg/kg/day
LOAEL = 800 mg/kg/day based on decreased body weights, and decreased hemoglobin-related parameters accompanied by hemosiderosis of the spleen, increased liver and spleen weights, and decreased thymus weights.
28-Day oral capsule dogs
44014231 (1994) 
Acceptable/nonguideline
0, 50, 250, 500 mg/kg/day
NOAEL = 50 mg/kg/day
LOAEL = 250 mg/kg/day based on decreased body weight, decreased hemoglobin-related parameters, hepatic and splenic hemosiderosis.
90-Day Dietary 
mice
44014228 (1993)
Acceptable/nonguideline
M: 0, 30.6, 152, 624 mg/kg/day; F: 0, 47.4, 220, 803 mg/kg/day
NOAEL =  M: 30.6 mg/kg/day; F: 47.4 mg/kg/day
LOAEL = M: 152 mg/kg/day; F: 220 mg/kg/day based on decreased mean body weights and body weight gain in males, increased spleen weights and splenic histopathology in both sexes. 
90-Day Dietary 
dog
44014232 (1994)
Acceptable/nonguideline
0, 10, 50, or 200 mg/kg/day
NOAEL =  50 mg/kg/day
LOAEL = 200 mg/kg/day in male and female dogs, based on regenerative hemolytic anemia evidenced by decreased erythroid parameters, pigmentation in the liver and spleen, splenic congestion, and by activation of medullary (bone marrow) and extramedullary (splenic) erythropoiesis.
90-Day Dietary 
rat
44014230 (1993)
Acceptable/nonguideline
males: 0,2.42, 24.6, 126, or 516 mg/kg/day; females: 0,2.64,26.3, 131, or 554 mg/kg/day)
NOAEL = 126 mg/kg/day in males and 131 mg/kg/day
in females 
LOAEL = 516 mg/kg/day males and 554 mg/kg/day females based on decreased mean body weights, reduced food intake, reduced food efficiency, and increased liver and spleen weights with correlates for the liver and spleen of glycogen deposition and hemosiderosis, respectively. 
Special
Developmental toxicity 
rats
44014238 (1994)
Acceptable/nonguideline
300 mg/kg/day, GD 6-15, 6-7, 8-9, 10-11, 12-13, or 14-15
Maternal and developmental NOAELS and LOAELS could not be identified by this protocol.  The most pronounced maternal and developmental toxicity occurred when dams were treated on GD 6-15.
Special
Developmental toxicity
rats
44014239 (1994)
Acceptable/nonguideline
400 mg/kg/day, GD 6-7, 8-9, 10-11, 12-13, or 14-15
Maternal and developmental NOAELS and LOAELS could not be identified by this protocol.  The most pronounced maternal and developmental toxicity occurred when dams were treated on GD 6-7 and 8-9.
Dermal developmental toxicity
rats
44014240 (1994)
Acceptable/nonguideline
0, 10, 100, 500 mg/kg/day, GD 6-15
Maternal NOAEL 500 mg/kg/day
LOAEL >500 mg/kg/day based on no effects.
Developmental NOAEL 500 mg/kg/day
LOAEL >500 mg/kg/day based on no effects.
Range-finding 
1-generation reproduction 
rats
44681301 (1993)
Acceptable/nonguideline
0, 199-209, 382-410, 700-728 mg/kg/day

Parental/Systemic NOAEL = 209 mg/kg/day
LOAEL = 410 mg/kg/day based on decreased body weight gain and food consumption in females.
Reproductive NOAEL = 410 mg/kg/day
LOAEL = 728 mg/kg/day based on total resorptions in all dams.
Offspring NOAEL = 209 mg/kg/day
LOAEL = 410 mg/kg/day based on reduced pup body weight gains and lower pup body weights during lactation.
A.3	Toxicological Endpoints

A.3.1	Acute Reference Dose (aRfD) - General Population
      
	Selected Study: Developmental Neurotoxicity in Rats (MRID 46046401).  
	See Section 3.2.3

	Dose and Endpoint for Establishing an aRfD:  8.2 mg/kg/day based on an offspring NOAEL of 8.2 mg/kg/day and an offspring LOAEL of 82 mg/kg/day based on changes in brain morphometrics in the cerebellum.  
      
      Uncertainty Factor (UF): 100  -  10x for interspecies extrapolation and 10x for intraspecies variation.
      
      Comments about Study/Endpoint:  The developmental neurological effects of concern are presumed to occur after a single exposure and are relevant to females aged 13-50 years old since they occur in utero and to young children based on post-natal effects.  
      
      [Note: In the previous risk assessment (D361893, 3/3/09) only the subpopulation females 13-50 years old was assessed for acute dietary exposure based on in utero effects.  However, based on a reevaluation of the DNT study regarding the potential for post-natal toxicity, the acute dietary analysis has been expanded to include children for this assessment.]
        
               Acute RfD  =    8.2 mg/kg (NOAEL) = 0.0.082 mg/kg/day
  (General Population)	           100 (UF)
      
      
      
      
A.3.2 	Chronic Reference Dose (cRfD) - Females age 13-49 and Young Children
      
      Selected Study: Developmental Neurotoxicity in Rats (MRID 46046401).  
      See Section 3.2.3

	Dose and Endpoint for Establishing a cRfD:  8.2 mg/kg/day based on an offspring NOAEL of 8.2 mg/kg/day and an offspring LOAEL of 82 mg/kg/day based on changes in brain morphometrics in the cerebellum.  
      
      Uncertainty Factor (UF): 100  -  10x for interspecies extrapolation and 10x for intraspecies variation.
      
      Comments about Study/Endpoint:  The effects observed in this study can occur after single or multiple doses.  Selection of a chronic endpoint from this study ensures that the chronic exposure assessment is protective of females of reproductive age and their offspring.  
      
      [Note: In the previous risk assessment (D361893, 3/3/09) only the subpopulation females 13-50 years old was assessed for chronic dietary exposure based on in utero effects observed in the DNT study.  However, based on a reevaluation of the DNT study regarding the potential for post-natal toxicity, the chronic dietary analysis the chronic dietary exposure analysis has been revised to include an assessment of dietary risks to children using the NOAEL from the DNT study.]
      
                       Chronic RfD  =               (NOAEL) 8.2 mg/kg =  0.082 
   (Females 13-49/Young Children)          (UF) 100	
      
      
      
      
A.3.3 	Chronic Reference Dose (RfD)  -  Adult Males and Females 50+ years

      Selected Studies: Co-critical studies Chronic Feeding Study in Dogs (MRID 44014234); Oncogenicity Study Mouse (MRID 44014235); Chronic/Oncogenicity Study Rat (MRID 44014243); Reproductive Study Rat (44014241)
      
      Chronic Dog Study - In a chronic oral toxicity study acibenzolar-S-methyl was administered to 4 beagle dogs per sex per dose level in gelatin capsules at levels of 0, 5, 25, or 200 mg/kg/day for 52 weeks.  No biologically significant toxic effects were observed in either males or females receiving 25 mg/kg/day or less of the test material. At 200 mg/kg/day, there was an indication of hemolytic anemia in dogs of both sexes i.e., effects consistent with prior or current hemolytic anemia including hematological effects, hemosiderosis of the liver and spleen, extraniedullary hematopoiesis in the spleen, and increases in liver weight.  Significant treatment-related changes were found in clinical chemistry in the dogs fed the test, substance at 200 mg/kg/day, (increase in triglycerides in both sexes, increase of cholesterol in females at week 13, decrease in total protein in males at weeks 26 and 52, and decrease in albumin in both sexes at week 13. These clinical chemistry changes are suggestive of possible nutritional deficiencies.  The LOAEL is 200 mg/kg/day in both sexes based on effects consistent with prior or current hemolytic anemia.  The NOAEL is 25 mg/kg/day.  
      
      Oncogenicity Mouse Study - In an oncogenicity study acibenzolar-S-methyl was administered in the diet to groups of 60 male and 60 female Tif:MAGf (SPF) mice at concentrations of 0, 10, 100, 2000, or 6000 ppm (1.14, 11.11, 237, and 698 mg/kg/day for males and 1.14, 10.8, 234, and 696 mg/kg/day for females) for 18 months.  Ten mice per group were reserved for measurements of hematology parameters after 12 and 18 months of treatment.  Treatment did not result in a significant decrease in survival.  The overall food efficiency was decreased in females by 22% at 6000 ppm.  The red blood cell count, hemoglobin, and hematocrit at 53 and 79 weeks were decreased in females at 2000 ppm and 6000 ppm.  Mean corpuscular volume was decreased in high dose males at 79 weeks.  Mean corpuscular hemoglobin concentration distribution increased at 6000 ppm.  Reticulocyte counts in high-dose females was significantly increased.  The hematology data are consistent with hemolytic anemia especially in females at 2000 and 6000 ppm.  Macroscopic and microscopic findings consistent with a mild hemolytic anemia in both sexes included increased incidences of enlarged spleen, increased incidences of hemosiderosis in liver, spleen, and bone marrow.  Incidence of extramedullary hematopoiesis in the spleen and pancreatic exocrine hyperplasia was increased in males at 2000 and 6000 ppm.  Incidences of alveolar foam cells in the lungs were increased at 6000 ppm in both sexes.  The LOAEL is 2000 ppm (237/234 mg/kg/day males/females), based on mild hemolytic anemia and related microscopic changes (hemosiderosis) in the spleen, liver, and bone marrow and extramedullary hematopoiesis in the spleen.  The NOAEL was 100 ppm (11.1 mg/kg/day males; 10.8 mg/kg/day females).
      
      Chronic/Oncogenicity Rat Study - In a chronic toxicity/oncogenicity study, acibenzolar-S-methyl was administered in the diet to groups of 80 male and 80 female (Tif:RAIf (SPF)) Sprague-Dawley derived rats at concentrations of 0, 20, 200, 2500, or 7500 ppm (0, 0.77, 7.77, 96.9, and 312 mg/kg/day male and 0, 0.90, 9.08, 111, and 388 mg/kg/day female).  Ten animals per sex per dose were sacrificed at 52 weeks for interim evaluation; the remaining animals were used for hematologic and clinical chemistry evaluation (20 per sex per group) and evaluation of carcinogenic potential (50 animals per sex per group).  No treatment-related effects were observed at 20, 200 or 2500 ppm for any parameter.  Treatment related effects on body weight, weight gain, food consumption, and food efficiency were observed in both sexes at 7500 ppm.  Other treatment-related effects at 7500 ppm included a slight hemolytic anemia in both sexes as evidenced by decreases in erythrocyte count, increased mean corpuscular volume, elevated reticulocyte counts, increased serum bilirubin, increased severity of hemosiderin deposition in the spleen, and increased incidence of hemosiderin deposition in Kupifer cells of the liver.  Hemosiderin deposition in the spleen was accompanied by a statistically significant increase in spleen weight.  There was also a statistically significant increase in the incidence of foam cells in the lungs of females fed the 7500-ppm diet.  The LOAEL was 7500 ppm (312/388 mg/kg/day males/females) based on decreased body weights, body weight gain and food efficiency, mild hemolytic anemia, and increased incidence of alveolar foam cells (females only). The NOAEL was 2500 ppm (96.9 mg/kg/day males and 111 mg/kg/day females).  

      Reproductive Study in Rats - See Section 3.2.2
      
      Dose and Endpoint for Establishing cRfD:  25 mg/kg/day based on a NOAEL of 25 mg/kg/day from the chronic dog study and a LOAEL of 105 mg/kg/day from the rat reproduction study based on hemolytic anemia with compensatory response. 
      
      Uncertainty Factor(s): 100  -  10x for interspecies extrapolation and 10x for intraspecies variation.

	Comments about Study/Endpoint/Uncertainty Factor: The NOAEL of 25 mg/kg/day, taken from the chronic dog study, is based on the totality of the database with a LOAEL of 105 mg/kg/day (the lowest LOAEL is from the reproduction study) based on hemolytic anemia with compensatory response.  The NOAEL and LOAEL recommended for use in the risk assessment come from different studies conducted in different species.  Typically, a combination of NOAELs/LOAELs from studies conducted in different species is not recommended.  However, in the case of acibenzolar-S-methyl the same endpoint (hemolytic anemia) is observed across species at a relatively close range of LOAELs (105-312 mg/kg/day).  Therefore, the ToxSAC concluded that, in this instance, it would be appropriate to base the point of departure for the risk assessment on the NOAEL from the dog chronic toxicity study using the LOAEL from the reproduction study in rats for the basis of the endpoint (hemolytic anemia).
      
            Chronic RfD  =    25 mg/kg/day (NOAEL) = 0.25 mg/kg/day
        (Adult Male)	           100 (UF)
	   
   
   	
   
A.3.4   	Incidental Oral Exposure (Short- and Intermediate-Term)
      
      Selected Study: Developmental Neurotoxicity in Rats (MRID 44014236).  
      See Section 3.2.3

	Dose and Endpoint:  8.2 mg/kg/day based on an offspring NOAEL of 8.2 mg/kg/day and an offspring LOAEL of 82 mg/kg/day based on changes in brain morphometrics in the cerebellum.  
      
      Uncertainty Factor (UF): 100  -  10x for interspecies extrapolation and 10x for intraspecies variation.
      
	Comments about Study/Endpoint:  This endpoint is considered appropriate based on the  post-natal component of the DNT.  

A.3.5	Dermal Absorption

	A dermal absorption factor (DAF) is applied when dermal exposure endpoints are selected from oral toxicity studies.  The dermal factor converts the oral dose to an equivalent dermal dose for the risk assessment.  A DAF of 45% was selected for use in risk assessment based on a dermal absorption study in rats.  

A.3.6	Dermal Exposure (Short and Intermediate-Term)

      Selected Study: Developmental Neurotoxicity in Rats (MRID 44014236).  
      See Section 3.2.3

	Dose and Endpoint:  8.2 mg/kg/day based on an offspring NOAEL of 8.2 mg/kg/day and an offspring LOAEL of 82 mg/kg/day based on changes in brain morphometrics in the cerebellum.  
      
      Uncertainty Factor (UF): 100  -  10x for interspecies extrapolation and 10x for intraspecies variation.
      
	Comments about Study/Endpoint:  Two acceptable dermal toxicity studies, including a dermal developmental study were available.  However since neurological effects observed in the oral developmental neurotoxicity study are not measured in the dermal developmental study, the findings from the dermal study may not be protective of these neurological effects.  Since an oral NOAEL was selected for dermal risk assessment, a dermal absorption factor of 45% should be used.  This factor is based on a dermal absorption study in rats.

A.3.7 	Inhalation Exposure (Short and Intermediate-Term)

      Selected Study: Developmental Neurotoxicity in Rats (MRID 44014236).  
      See Section 3.2.3

	Dose and Endpoint:  8.2 mg/kg/day based on an offspring NOAEL of 8.2 mg/kg/day and an offspring LOAEL of 82 mg/kg/day based on changes in brain morphometrics in the cerebellum.  
      
      Uncertainty Factor (UF): 100  -  10x for interspecies extrapolation and 10x for intraspecies variation.
      
	Comments about Study/Endpoint:  Since an oral NOAEL was selected for inhalation risk assessment, an inhalation absorption factor of 100% should be used



A.4		EXECUTIVE SUMMARIES 

870.3700  Developmental Toxicity

In a non-guideline developmental toxicity study (MRID 44014238), twelve presumed pregnant Tif:RAT f(SPF) (hybrids of RTT/1 x RII/2) rats per group were administered CGA 245704 Technical (97.9%; Batch No. P.303011) in a 0.5% aqueous solution of sodium carboxymethylcellulose by gavage at a dose of 300 mg/kg/day. Groups 2-7 were administered test article on gestation days (GD) 6-15,6- 7, 8-9, 10-11, 12-13, or 14-15, respectively. Group 1 consisted of twelve animals which served as controls and were treated with 0.5% sodium carboxymethylcellulose on GD 6-15. On GD 21, dams were sacrificed, subjected to gross necropsy, and all fetuses examined externally. Visceral and skeletal examinations were not performed.  All animals survived until scheduled sacrifice on GD 21. Bloody, vaginal discharge was observed in 8/12 dams in Group 2, 3/12 dams in Group 3, 5/12 dams in Group 4, 4/12 dams in Group 5, and in 5/12 dams in Group 6. This finding was observed for one to four days starting on GD 13 and was not observed in controls or in animals in Group 7. No other treatment-related clinical signs of toxicity were observed. No statistically significant differences in absolute body weights or body weight changes occurred at any time between the treated groups and the control group. Body weight gain by Group 2 was 80% of the control level during the treatment interval (GD 6- 16), with the greatest reduction in weight gain (77% of controls) during GD 11-16. Food consumption was not affected. There were no statistically significant differences between control and treatment groups for number of corpora lutea, number of implantation sites, live fetuses/dam, pre- and post-implantation loss, or fetal sex ratios. Fetal body weights from the Group 2 dams were significantly (p <0.01) less than the controls. Although not statistically significant, Group 2 dams also had fewer live fetuses per litter (10.9 vs 14.8 for controls) and a greater post implantation loss (30.1% vs 2.9% for controls) from early resorptions due to one dam with whole liner resorption (17/17) and 2 additional dams with 13/16 and 8/9 resorptions/number of implantation sites. The overall incidence rates for litters containing fetuses with major external malformations in Groups 1-7 were 0/12, 1/10, 0/10, 1/11, 0/11, 0/10, and 0/12, respectively. In one Group 2 litter, generalized edema was observed in 5/14 fetuses. In addition, two of the five also had a position anomaly of the hindlimb and one of the two also had gastroschisis. These five fetuses also had greatly reduced body weights. Generalized edema was also seen in one fetus from Group 4.  Maternal and developmental toxicity NOAELs and LOAELs could not be identified by this protocol, which investigated the critical period of organogenesis. The most pronounced maternal and developmental toxicity occurred when dams were treated on GD 6-15, inclusive at a dose level of 300 mg/kg/day. This study is classified as Acceptable/non-guideline as a developmental toxicity study (83-3a) in rats.

In a non-guideline developmental toxicity study (MRID 44014239), eight presumed pregnant Tif: RAT f (SPF) (hybrids of RII/1 x RII2) rats per group were administered CGA 245704 Technical (97.9%; Batch No. P.303011) by gavage at a dose of 400 mg/kg/day. Groups 2-6 were administered test article on gestation days (GD) 6-7, 8-9, 10-11, 12-13, or 14-15, respectively. Group 1 consisted of eight animals which served as controls and were treated with 0.5% carboxymethylceliulose (vehicle) on GD 6-15. On GD 21, dams were sacrificed, subjected to gross necropsy, and all fetuses examined externally. Visceral and skeletal examinations were not performed.  One Group 2 dam was found dead on GD 20. Prior to death, this animal had bloody perineal discharge and necropsy findings including mottled kidney and thymus. One Group 4 dam delivered on GD 20 and was sacrificed. All other animals survived until scheduled sacrifice on GD 21. Bloody, perineal discharge was observed in 4/8 dams in Group 3. This finding was observed for three to four days starting on GD 16. The same 4 dams were later observed to have total lifter resorptions. No other treatment-related clinical signs of toxicity were observed. No statistically significant differences in body weights, body weight gains, or food consumption occurred at any time between the treated groups and the control group.  There were no differences between the control group and Groups 4, 5, and 6 for number of corpora lutea, number of implantation sites, live fetuses/dam, pre- and post-implantation loss, fetal body weights, or fetal sex ratios. Group 2 had a slightly greater number of early resorptions/dam (2.7 vs 0.9 for controls) and an increased postimplantation loss (18.7% vs 5.4% for controls). The four Group 3 dams observed with bloody discharge had complete litter resorption resulting in increased postimplantation loss (53.4%). Group 3 also had an increase in early rcsorptions (8.0/dam vs 0.9/dam for controls) and decreased mean live fetuses/litter (8.5 vs 16.0 for controls). Another Group 3 dam had fused placenta between two fetuses.    The overall incidence rates for liners containing fetuses with major external malformations in Groups 1-6 were 0/8, 0/6, 0/4, 1/7, 0/8, and 0/8, respectively. One Group 4 fetus had cleft palate and another fetus from the same lifter had a kinked tail. No treatment-related external malformations /variations were observed in any fetus from any group. Maternal and developmental toxicity NOAELs and LOAELs could not be identified by this protocol, which investigated the critical period of organogenesis. Pronounced maternal and developmental toxicity occurred when dams were treated on GD 6-7 and 8-9 at a dose level of 400 mg/kg/day.

870.7600 Dermal Toxicity Studies

In a dermal developmental toxicity study (MRID 44014240), 24 pregnant Tif: RAIf (SPF) rats (Sprague-Dawley derived) per group were administered acibenzolar-S-methyl (97.9% a.i.) in aqueous carboxymethylcellulose (0.5% w/w) in 0.1% aqueous Polysorbate 80, by dermal application at doses of 0 (vehicle), 10, 100, or 500 mg/kg/day on gestation days (GD) 6-15, inclusive.  Exposure duration was 6 hours per day. On GD 21, all dams were sacrificed and all fetuses were sexed, weighed, and examined for external malformations/variations, then sacrificed. Approximately one-half of each litter was placed in Bouin's fixative for subsequent visceral examination and the remainder were prepared and stained for skeletal examination.  All animals survived until scheduled sacrifice.  No clinical signs of toxicity were observed at any treatment level.  Gestational body weights, body weight changes, and food consumption were not statistically significantly different for any treatment level throughout the gestational period.
Therefore, the maternal toxicity NOAEL is 500 mg/kg/day based on no adverse maternal effects at the highest administered dose.  The maternal toxicity LOAEL was >500 mg/kg/day.  No treatment-related effects were observed for gravid uterine weights, number of fetuses/litter, pre- and post-implantation losses, numbers of corpora lutea/dam, number of implantations/dam, resorptions/dam, fetal body weights, or fetal sex ratios.  No biologically relevant statistically significant differences in the incidence rates of any external, visceral, or skeletal malformations/ anomalies/variations were observed in the treated litters as compared to the controls.  There were statistically significantly decreased incidences of absent ossification of the proximal phalanx of anterior digit-2 in all treatment groups and poor ossification of the proximal phalanx of posterior digit-2 in the 500 mg/kg/day treatment group as compared to the control group. However, these decreases were not considered to be biologically significant.  Therefore, the developmental toxicity NOAEL is 500 mgfkglday based on no adverse developmental effects at the highest dose administered. The developmental toxicity LOAEL was >500 mg/kg/day.

In a 28- day repeated dose dermal toxicity study (MRID 44014233), groups of 5 females Tif: RAIf (SPF) albino rats were treated with acibenzolar-S-methyl technical in 0.5% (w/v) carboxymethyl cellulose/0.1% (w/v) aqueous polysobate 80 at doses of 0, 1, 10, 100 or 1000 mg/kg/day.  Animals were treated by dermal occlusion for 6 hours/day, 5 days/week for 4 weeks.  No animals died during the study.  There were no treatment- related clinical signs, dermal effects, effects on body weight, food consumption, hematology, clinical chemistry, or organ weight.  No gross or microscopic pathology were noted at necropsy.  The systemic and dermal NOAEL is the limit dose of 1000 mg/kg/day.  A dermal and systemic LOAEL were not identified.  

870.3800 Reproduction

In a 1-generation range-finding reproduction study in rats (Nonguideline study, MRID 44014241) CGA 245704 Technical (97.9% a.i.; Batch No. P.303011) was administered to groups of 15 male and 15 female Tif: RAT f (SPF), hybrids of RTTI 1 x P11/2 (Sprague-Dawley derived) rats at dietary concentrations of 0, 2000, 4000, or 8000 ppm for one generation (MRID 44681301). In this range- finding study, F0 adults were administered test or control diet for two weeks prior to mating. Premating doses were 199.4, 382.0, and 700.1 mg/kg/day, respectively, for F0 males and 208.7, 409.9, and 727.8 mg/kg/day, respectively, for F0 females. Males were terminated at the end of mating and females were terminated at weaning of their pups. All offspring, except five litters per dose group, were necropsied at weaning. After weaning, five litters per dose group were retained on treatment for an additional three weeks and then necropsied.  The only clinical sign of toxicity in the parental animals was perineal hemorrhage during late gestation in five high-dose females. Gross necropsy was unremarkable. Absolute body weights, body weight gains, and food consumption values for the low- and mid-dose males and low-dose females were similar to the controls throughout the study. High-dose males had significantly (p > 0.01; 88-91% of control) lower body weights than the controls beginning on day 8 and continuing until study termination. Overall weight gain by the high-dose males was 69% of the control level. Body weights and body weight gains of the high-dose females were significantly (p > 0.01; body weights 88-90% of controls) Tess than the controls during the premating interval. Overall body weight gain by the high- dose group was only 30% of the control group level. Mid-dose females had significantly (p > 0.05) lower body weight gains than the controls during days 1-8. Reduced body weight gains in the high- dose males and females and mid-dose females correlated with significantly (p > 0.05 or 0.01) reduced food consumption by these treated groups during study days 1-8. Lower body weights, body weight gains, and food consumption continued for the mid- and high-dose females during gestation and lactation. Body weights of the high-dose group were significantly (p > 0.05 or 0.01) lower than the controls throughout gestation with overall weight gain by the high-dose females during gestation 27% (p > 0.01) of the control group level. Mid-dose females had significantly (p > 0.05 or 0.01) lower body weights than the controls on GD 21 and on lactation days 7, 14, and 21. Weight gains by the mid-dose group during gestation and lactation were 92% and 83%, respectively of the control group levels. Food consumption by the high-dose group was significantly (p > 0.05 or 0.01) less than the controls throughout gestation. Food consumption by the mid-dose group was significantly (p > 0.01) less than the controls throughout lactation. Therefore, the LOAEL for parental toxicity is 4000 ppm based on reduced food consumption and lower body weight gains in F0 females. The parental toxicity NOAEL is 2000 ppm.  Mating and fertility indices, mean precoital interval, mean gestation length, and mean numbers of corpora lutea and implantation sites per dam were similar between the treated and control groups. Mating and fertility indices for the high-dose group were similar to the control group, but no live lifters were produced by the high-dose group (i.e. all pregnancies were lost). Live births and pup survival were similar between the low- and mid-dose groups and the control group. Therefore, the LOAEL for reproductive toxicity is 8000 ppm based on total resorptions in all dams. The reproductive toxicity NOAEL is 4000 ppm.  No treatment-related clinical signs of toxicity were observed in the pups during lactation. Absolute body weights of pups in the low-dose group were similar to the controls throughout lactation. Body weights of the mid-dose pups were significantly (p > 0.05 or 0.01; 76-89% of controls) less than the controls beginning on lactation day 7 and continuing throughout lactation and postweaning. The lower body weights of the mid-dose pups were a result of significantly (p > 0.05 or 0.01; 69-89% of controls) lower body weight gains from postnatal day 4 through postnatal day 28. Recovery of body weight gains occurred during the postweaning intervals of days 28-35 and 35-42. Therefore, the LOAEL for developmental toxicity is 4000 ppm based on reduced pup body weight gains and lower pup body weights during lactation and early postweaning. The developmental toxicity NOAEL is 2000 ppm.

870.6200b Subchronic Neurotoxicity

In a subchronic neurotoxicity study (MRID 45713601), CGA-245704 (Acibenzolar, 97.9% a.i., Batch No. P.303011) was administered in the diet to 10 Sprague-Dawley derived rats/sex/group at dose levels of 0, 400, 2000, or 8000 ppm (equivalent to 0/0, 24/26, 126/143, and 575/628 mg/kg/day [M/F], respectively) for at least 90 days.  Neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) was performed using all rats at pre-dosing and Weeks 4, 8, and 13.  At study termination, 5 rats/sex/group were anesthetized and perfused in situ for neuropathological examination.  The tissues from the perfused animals in the control and 8000 ppm groups were subjected to histopathological evaluation of brain and peripheral nervous system tissues.  Positive control data were not provided.No compound-related effects were observed in mortality, clinical signs of toxicity, FOB, motor activity, or gross or neuropathology.  At 8000 ppm, body weights were generally decreased throughout the study in the males (decreased 11-14%, not statistically significant) and females (decreased 8-13%, attaining statistical significance at Weeks 2-5, 7 and 8, and 11-13).  Also at this dose, cumulative body weight gains were decreased throughout the study in the males (decreased 19-35%, attaining statistical significance at Weeks 2-4 and 6-9), and in the females (decreased 24-40%).  A decreasing trend (p<0.01) in body weight and body weight gain was also generally noted in both sexes at this dose.  These decreases in body weight gain corresponded with decreases in food consumption which was sporadically decreased in the 8000 ppm males (decreased 9-21% at Weeks 1-3, 5-7, and 11, but only attaining statistical significance during Week 1), and females (decreased 14-18%, p<0.01 at Weeks 1 and 3).  A decreasing trend (p<0.01) in food consumption was also generally noted in the males at 8000 ppm during the weeks reported above. No neurological effects were observed at any dose in either sex.

The LOAEL was 8000 ppm (equivalent to 575/628 mg/kg/day, M/F) based on decreased body weights, body weight gains, and food consumption.  The NOAEL is 2000 ppm (equivalent to 126/143 mg/kg/day, M/F).

The subchronic neurotoxicity study in rats (MRID 45713601) on Acibenzolar had been reviewed and is classified as acceptable/non-guideline. 

870.5100, 870-5300, 870.5375, 870.5900  Mutagenicity 

Five acceptable mutagenicity studies on technical grade acibenzolar-S-methyl are available:

1. 	Reverse gene mutation, S. typhimuriumlE. Coli (MRID 44014247)
2. 	Forward gene mutation, Chinese hamster V79 lung cells in culture/HGPRT locus (MRID
	44014246)
3. 	Chromosome aberration, Chinese hamster ovary (CHO) cells in culture (MRID 44014245)
4. 	Unscheduled DNA synthesis assay, primary rat hepatocytes in culture (MRID 44014248)
5. 	In vivo cytogenetics, micronucleus assay in mice (MRID 44014244)
	Results in all five studies were negative for genotoxicity. The five studies on the technical grade product satisf' the new revised mutagenicity guideline requirements for a new chemical (published in 1991). 

Three additional acceptable mutagenicity studies on a) an isomer (CGA-362020) of acibenzolar S-methyl, b) a by-product (NOA 419191) of acibenzolar-S-methyl, and c) a plain metabolite (CGA-323060) of acibenzolar-S-methyl are also available.

1. 	Reverse gene mutation, S. typhimuriumlE. Coli; Test Material: CGA-362020 (MRID
	44537025). This study was POSITIVE for evidence of induced mutant colonies over background in S. typhimurium strain TA1537 at concentrations of 277.8 and higher in the absence of 59-mix.
2. 	Reverse gene mutation, S. typhimuriumlE. Coli; Test Material: NOA 419191 (MRID
	44537024). This study was negative.
3. 	Reverse gene mutation, S. typhimuriumfE. Coli; Test Material: CGA-323060 (MRlD
	44537026). This study was negative

CGA-362020, an isomer of acibenzolar-S-methyl, is a low level impurity arising from a new production process for acibenzolar-S-methyl, which has not yet been submitted to the Agency. Trace levels of this isomer (<0.1%) were found in some batches of technical grade acibenzolar-S-niethyl produced via the new production process. See the 6(a)(2) letter from Novartis, dated 3/16/98, reporting the findings in this mutagenicity study and the above information regarding the formation of this isomer in the new production process. Presumably, the same isomer does not occur in the old production process which produced the test material (lot/batch # P.303011) that was used for virtually all the toxicity testing on acibenzolar-S-methyl up to this point in time.
APPENDIX B.  Chemical Names And Structures Of Metabolites









APPENDIX C. Physical/Chemical Properties

Physicochemical Properties of Acibenzolar-S-Methyl.
Parameter
Value
Reference
Melting point/range
133 ºC
DP# 250963, 2/10/99, H. Podall
pH
7.9 (1% solution in water) at 25 ºC

Density
1.54 g/cc at 22 ºC (bulk density)

Water solubility
0.0077 g/L

Solvent solubility
Methanol	  4.2 g/L
Acetone		  28 g/L
Toluene		  36 g/L
n-Octanol	  5.4 g/L
n-Hexane	  1.3 g/L
Ethyl acetate	   25 g/L
Dichloromethane   160 g/L 
European Commission, 6506/VI/99-final, 5/21/02
Vapor pressure
3.6 x 10[-6] Torr at 25 ºC
DP# 250963, 2/10/99, H. Podall
Dissociation constant, pKa
No dissociation constant in an
accessible pH range (1.0 - 9.0)
European Commission, 6506/VI/99-final, 5/21/02
Octanol/water partition coefficient, Log(KOW)
Log (POW) 3.1 at 25 ºC
DP# 250963, 2/10/99, H. Podall
UV/visible absorption spectrum
253 nm ε : 14.7 10[3] l mol[-1] cm[-1]
288 nm ε : 5.4 10[3]
324 nm ε : 7.2 10[3]
European Commission, 6506/VI/99-final, 5/21/02

APPENDIX D.  Studies Reviewed for Ethical Conduct

The PHED Task Force, 1995.  The Pesticide Handlers Exposure Database, Version 1.1.  Task Force members Health Canada, U.S. Environmental Protection Agency, and the National Agricultural Chemicals Association, released February, 1995.

Agricultural Re-entry Task Force (ARTF) data base (SOP #3.1)


