Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 1 of 52 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460 OFFICE OF C HEMICAL SAFETY AND POLLUTION PREVENTION **MEMORANDUM** March 3 , 2015 **SUBJECT: Benalaxyl**  **- M.**  Human - Health Risk Assessment for Tolerances in/on Imported

Grape and Tomato . **PC Code:** 113510 **DP Barcode:** D 416044 **Decision No.:** 476296 **Registration No. :**  None **Petition No.:** 3 E 8162 **Regulatory Action:** Tolerance , No U.S. Registration **Risk Assessment Type:** Single Chemical **Case No.:** NA **T XR No.:**  NA **CAS No.:** 688046 - 61 - 9 **MRID No.:** NA **40 CFR:** 180.TBD **FROM:** William Donovan , Ph.D., Chemist/Risk Assessor

Linda Taylor, Ph.D., Senior Toxicologist Ris k Assessment Branch V/VII Health Effects Division (HED) (7509P) **THROUGH:** Michael Metzger , Branch Chief

Risk Assessment Branch V/V II , HED (7509P) Anna Lowit, Senior Toxicologist Immediate Office, HED (7509P) Christine Olinger, Branch Chief Risk Assessment Branch II I, HED (7509P) **TO:** B e wanda Alexander , Risk Manager

Rach el Holloman, Branch Chief Fungicide/Herbicide Branch Registration Division (7505P) The Health Effects Division (HED) of the Office of Pesticide Programs (OPP) is charged with estimating the risk to human health from exposure to pesticides. The Registration Division (RD) of OPP has requested that HED evaluate hazard and exposure data and conduct assessments, as needed, to estimate the risk to human health that will result from the proposed uses of the new fungicide active ingredient benalax yl - M .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 2 of 52 HED has evaluated the toxicity and exposure databases for the new active ingredient, benalaxyl M, and has conducted a human health risk assessment in support of the proposed uses. Based on this assessment, HED has determined that there are no potential risk estimates of concern for the proposed uses of benalaxyl - M. HED recommends that the requ ested uses be granted. A summary

of the findings and an assessment of human health risk resulting from the proposed uses for benalaxyl - M are provided in this document. The HED team members contributing to this risk assessment include Li nda Taylor (hazard evaluation) and William Donovan ( chemistry , dietary , and risk assessment s ).Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 3 of 52 

### Table of **Contents** 1.0 Executive Summary ................................ ................................ ................................ ............. 5 2.0 HED Recommendations ................................ ................................ ................................ ....... 6 2.1 Data Deficiencies ................................ ................................ ................................ ............. 6 2.1.1 Toxicity

Studies ................................ ................................ ................................ ............ 6 2.2 Tolerance Considerations ................................ ................................ ................................ . 7 2.2.1 Enforcement Anal yt ical Method ................................ ................................ .................. 7 2.2.2 International Harmonization ................................ ................................ ......................... 7 2.2.3 Recommended Tolerances ................................ ................................ ............................ 7 2.2.4 Revisions to Petitioned - For Tolerances ................................ ................................ ........ 8 3.0 In gredient Profile ................................ ................................ ................................ ................. 8 3.1 Chemical Identity ................................ ................................ ................................ ............. 8 3.2 Phy sical/Chemical Characteristics ................................ ................................ ................... 8 3.3 Pesticide Use Pattern ................................ ................................ ................................ ........ 8 3.4 Anticipated Exposure Pathway s ................................ ................................ ..................... 10 3.5 Consideration of Environmental Justice ................................ ................................ ........ 10 4.0 Hazard Characterization and Dose - Response Assessment ................................ ................ 10 4.1 Toxicology

Studies Available for Analy sis ................................ ................................ ... 10 4.2 Absorption, Distribution, Metabolism and Elimination (ADME) ................................ . 11 4.2.1 Dermal Absorption ................................ ................................ ................................ ..... 11 4.3 Toxicological Effects ................................ ................................ ................................ ..... 12 4.4.1 Completeness of the Toxicology Database ................................ ................................ . 13 4.4.2 Evidence of Neurotoxicity ................................ ................................ .......................... 13 4.4.3 Evidence of Sensitivity /Susceptibility

in the Developing or Young Animal ............. 13 4.4.4 Residual Uncertainty in the Exposure Database ................................ ......................... 13 4.5 Toxicity

Endpoints and Points of Departure Selections ................................ ................. 14 4.5.1 Dose - Response Assessment ................................ ................................ ......................... 14 4.5.2 Recommendation for Combining Routes of Exposure for Risk Assessment .............. 14 4.5.3 Cancer Classification and Risk Assessment Recommendations ................................ . 14 4.5.4 Summary

of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment ................................ ................................ ................................ ................................ 15 5.0 Dietary

Exposure and 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 Pathway s ................................ ................................ ........... 16 5.1.3 Residues of Concern Summary and Rationale ................................ ........................... 16 5.2 Food Residue Profile ................................ ................................ ................................ ...... 17 5.3 Water Residue Profile ................................ ................................ ................................ .... 18 5.4 Dietary

Risk Assessment ................................ ................................ ................................ 18 5.4.1 Description of Residue Data Used in Dietary Assessment ................................ ......... 18 5.4.2 Percent Crop Treated Used in Dietary Assessment ................................ .................... 18 5.4.3 Acute Dietary

Risk Assessment ................................ ................................ ................. 18 5.4.4 Chronic Dietary

Risk Assessment ................................ ................................ .............. 18 5.4.5 Cancer Dietary

Risk Assessment ................................ ................................ ................ 18 5.4.6 Summary

Table ................................ ................................ ................................ ........... 19 6.0 Residential (Non - Occupational) Exposure/Risk Characterization ................................ .... 19Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 4 of 52 7.0 Aggregate Exposure/Risk Characterization ................................ ................................ ....... 19 8.0 Cumulative Exposure/Risk Characterization ................................ ................................ ..... 19 9.0 Occupational Exposure/Risk Characterization ................................ ................................ .. 20 10.0 References ................................ ................................ ................................ .......................... 20 Appendix A. Toxicology Profile and Executive Summaries ................................ ....................... 21 A.1 Toxicology

Data Requirements ................................ ................................ ...................... 21 A.2 Toxicity

Profiles ................................ ................................ ................................ ............. 22 A.3 Hazard Identification & Endpoint Selection ................................ ................................ .. 28 A.4 Executive Summaries ................................ ................................ ................................ ..... 28 Appendix B. Metabolism ................................ ................................ ................................ ............ 47 B.1 Metabolism Summary Table ................................ ................................ .......................... 47 B.2 Metabolic Pathway s ................................ ................................ ................................ ....... 50 Appendix C. Physical/Chemical Properties ................................ ................................ ................. 52 Appendix D. Review of Human Research ................................ ................................ ................... 52Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 5 of 52 **1 .0**  **Executive Summary**  Benalax yl - M is a new fungicide that is proposed for tolerances without U.S. registrations on grapes and tomatoes, with foliar application use proposed for these commodities. The end- use products include water- dispersible granules and wettable powders, where benalaxyl - M is co formulated with another active ingredient (mancozeb, folpet, or chlorothalonil). These fungicide formulations are intended to control downy mildew ( Plasmopara vitacola ) in grapes and late blight ( Phytophthora infestans) in tomato. The database of experimental toxicology studies available for benalax yl - M provides a robust characterization of the hazard potential of this pesticide. A comparative thyroid assay in rats is required to assess the potential impact of benalax yl - M exposure on thy roid function in the young given the pivotal role of thy roid hormones in brain development. The liver and th yroid are the primary target organs for benalaxyl - M . In rats, increased liver weights, clinical chemistry changes indicative of liver toxicity, hepatocellular hy pertrophy , and thy roid follicular cell hypertrophy were seen following subchronic and chronic exposure. In mice, increased liver weight and microsc opic lesions in the liver (hepatocellular hy pertrophy , necrosis, eosinophilic foci) were observed following subchronic and chronic exposure . Additionally , chronic exposure in rats and mice led to increases in the incidence of liver (rat, mouse) and th yroid (rat) tumors. Indogs, increased liver weight, changes in clinical chemistry indicative of liver toxicity, and hepatocellular hyp ertroph y were observed following subchronic exposure via the diet , whereas clinical chemistry changes indicative of liver tox icity, fat vacuolation in the liver, and thy roid follicular cell hy

pertrophy were observed following chronic exposure via capsules . No evidence of increased quantitative or qualitative susceptibility was seen in the benalax yl - M hazard database following in utero exposure with rats or rabbits in the prenatal developmental studies or in young rats in the 2

–generation reproduction study . No evidence of maternal toxicity or developmental effects w as observed in the developmental toxicity studies in rabbits or rats. T here is no reproductive concern. No neurotoxic effects were observed in the acute and subchronic neurotoxicity studies in rats , and no immunotoxic effects were observed in the immunotoxicity study in rats. Benalax yl - M was classified as “Likel y to be Carcinogenic to Humans”. This determination was based on the treatment - related liver tumors observed in male mice, liver tumors observed in male and female rats; and thy roid follicular cell tumors observed in female rats . No treatment - related tumors were observed in female mice. A linear low - dose extrapolation model (Q* 1 ) was used to estimate cancer risk, based on the male mouse liver tumor rates. There is no mutagenicity concern from the in vivo or in vitro genetic toxicity assay s. T oxicological endpoints were selected for dietary exposure scenarios onl y . An acute dietary endpoint was not selected because toxicity from a single exposure was not identified in the b enalax yl - M database. The chronic population adjusted dose ( c PAD ) is derived from the chronic toxicity / carcinogenicity s tudy

in rats , which had a lowest observed adverse effect level ( LOAEL ) of 13 5 mg/kg/day based on an increase in gamma glutam yltransferase ( GGT ) in males, increased trigl yc erides in males, increased cholesterol in both sexes, slight increases liver weight in both sexes, increased incidence of hepatocellular hy pertrophy in both sexes, increased incidence of th yr oid follicular cell hy pertrophy in both sexes, increased incidence of thy roid cellBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 6 of 52 hy perplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hy perplasia in females and a no observed adverse effect level (NOAEL ) of 20 mg/kg/day . For the point of departure (POD) for the chronic dietary ri sk assessment, a total uncertainty factor of 1000X was applied (10X for interspecies variability, 10X for intraspecies variability , and 10X for database uncertainty to account for the lack of a comparative th yroid assay). A full 10X factor was retained f or interspecies variability since other effects not linked to thy roid perturbations were also the basis of the POD. The residue chemistry

database is complete for the proposed uses, supporting a parent onl y resi due definition for tolerance enforcement and risk assessment. Based on the use of the Organization for Economic Cooperation and Development (OECD) Maximum Residue L imit (MRL) calculation procedures with residues reflecting critical Good Agricultural Pract ice (cGAP) use patterns, the recommended tolerance levels are 3 .0 and 0.2 0 ppm for grape and tomato, respectivel y. A risk assessment was conducted for dietary exposure only

based on the request for establishment of tolerances for import uses in/ on grape s and tomatoes . There are no drinking water, occupational or residential exposure assessments completed for non - domestic uses. Chronic dietary

analy ses were performed using conservative assumptions ( i.e ., 100% crop treated and mean field trial residue l evels ). There is no U.S. drinking water exposure associated with the establishment of an import tolerance; therefore, the chronic dietary exposure and risk assessments include food residues onl y. The resulting chronic exposure estimates were less than HE D’s level of concern ( ≤100% chronic PAD) for general U.S. population ( 1.4 % cPAD) and all population sub - groups. The most highly exposed population subgroup was Children 1 - 2 years old with an estimated risk of 7.1 % cPAD. The cancer risk estimate for the general U.S. population is 2 × 10 - 6

. This risk assessment does not rely on data from studies in which human subjects were intentionally

exposed to a pesticide or other chemical. **2.0 HED**  **Recommendations** Provided that benalax yl - M reference standard is submitted to the National Pesticides Standards Repository , there are no data deficiencies that would preclude establishing the recommended tolerances for benalaxyl - M in/ on grape and tomato . The specific tolerance recommendations are provided in Section 2. 2.3. **2.1 Data Deficiencies**  **2.1.1 Toxicity Studies**  HED has determined that a comparative thy roid assay

in rats is required to generate specific data on the thy roid hormone levels to protect the developing nervous sy stem from thy roid hormone disrupting chemicals. In the absence of th e s e data, the 10X FQPA database uncertainty factor has been retained in this risk assessment for the uncertainty associated with the potential for enhanced sensitivity

of the developing organism to prenatal or postnatal thy roid toxicity . ThisBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 7 of 52 uncertainty may be addressed by sub mission of a comparative thy roid assay or a proposal describing an alternative approach that adequatel y addresses the issue of disruption of thy roid hormone homeostasis during development. **2.2 Tolerance Considerations**  **2.2.1 Enforcement Analytical Method**  An adequate anal yt ical method is available to enforce the proposed tolerances for residues of benalax yl - M in plant commodities. Method RA.09.01, a high - performance liquid chromatograph y m ethod with tandem mass spectrometry detection (HPLC/MS/MS), has bee n adequatel y validated and radiovalidated, and has undergone a successful ILV (independent laboratory

validation). The method has limits of detection and quanti t ation of 0.005 and 0.010 ppm, respectivel y. Benalax yl - M has been tested using the Food and Drug Administration (FDA) multi - residue method (MRM) protocols. Complete recovery of benalax yl - M from tomato puree samples indicates that Protocol D of FDA Pesticide Anal ytical Methods (PAM) Vol. 1 may be applicable for determination of benalax yl - M. **2.2. 2**  **International Harmonization** Organization for Economic Cooperation and Development (OECD) Maximum Residue L imit (MRL) calculation procedures g ive rounded MRL estimates of 3 and 0.2 ppm for grape and tomato, respectivel y . Codex has set benalaxyl MRL s of 0.3 and 0.2 mg/kg for grape and tomato, respectivel y. Thus, the HED recommendations in this memo will result in harmonization of the U.S. tolerance with the Codex MRLfor tomato, but not for grape since benalaxyl - M residues from the grape trials in Argentina were significantly higher than the Codex MRL . **2.2.3 Recommended Tolerances**  HED has reviewed the available residue data and determined the appropriate tolerance levels for residues of benalax yl - M (Table 2.2.3). The recommended tolerance expression is as follows: Tolerances are established for residues of the fungicide benalax yl - M, 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 b y measuring only benalax yl[methyl N - (2,6 - dimethylphenyl ) -N - (phenylacetyl ) - DL - alaninate] in or on the commodity . **Table 2.2.**  **3 . Tolerance Summary for**  **Benalaxyl -**  **M .**  **Comm odity**  **Proposed Tolerance (ppm)**  **HED -**  **Recommended**

**Tolerance (ppm) Comments**  **;**

**Correct Commodit**

**y D efinition**  Grape 1.1 3 .0 Grape Grape, raisin 2.2 None Covered by grape tolerance Tomato 0.25 0.2 0 TomatoBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 8 of 52 The tolerance regulation should include a footnote to the tolerance stating : “There is no U.S. registration for use of benalax yl - M on grape or tomato .” **2.2.4 Revisions to Petitioned**  **- For Tolerances**  The proposed tolerance levels differ from those being recommended by HED. The petitioner used the NAFTA calculator to determine proposed tolerance levels while HED used OECD MRL calculation procedures. Further, for determination of the grape and tomato tolerance levels, the petitioner included the results from all trials. I n contrast, HED included only those data which matched the critical Good Agricultural Practice ( c GAP ) . **3.0 Ingredient**  **Profile 3.1**  **Chemical Identity TABLE 3.1.**  **Test Compound Nomenclature.** Compound

Common name Benalaxyl - M Company experimental name IR6141 and IR6141 M IUPAC name methyl N - (phenylacetyl) -N

- (2,6 - xyl yl) - D - alaninate CAS name methyl N - (2,6 - dimethylphenyl) -N

- (phenylacetyl) - D - alaninate CAS # 98243 - 83 - 5 End -use product/EP Fantic, Sidecar **3.2 Physical/Chemical Characteristics**  The physical and chemical properties of benalax yl - M are provided in Appendix C. Technical grade benalaxyl - M is a solid at room and biologically - relevant temperatures. The compound has low volatility

with little likelihood for exposure in the vapor phase. The technical - grade material has low aqueous solubili ty and a log Kow of 3.5 , indicative of potential for being a fat - soluble compound. **3.3 Pesticide Use Pattern**  The petitioner provided translations of the benalaxyl - M labels from Europe, Mexico, and Argentina. Table 3.3 summarizes the key use parameters for benalaxy l - M application to grape and tomato.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 9 of 52 **Table 3**  **. 3.**  **Summary of Directions for Use of Benalaxyl**  **- M.**  Country Formulation Application Type Application

Rate (kg ai/ha) Max. No.

Applic. per Season RTI 1

(days) Max.

Seasonal Applic. Rate (kg ai/ha) PHI 1

(days) Limitations Grape Italy Fantic M NC WG; Fantic M blu; [WP] Fantic F WG

Foliar 0.10 0.10 0.075 3

3 3 10 - 14 10 - 14 10 - 14 0.30

0.30 0.225 42

42 42 Apply the first treatment at the beginning of flowering (BBCH 60). Apply each subsequent treatment at an interval roughly coincident with the phenological stages of fully flowering (BBCH 65) and early development of the bunches (BBCH 73), when the berries reach the pea size. France Fantic F WG Sidecar [WP] 0.10 0.10 3

3 12 - 14 10 - 14 0.30

0.30 42

42 For table grapes, do not treat after flowering (Stage BBCH69) Spain Fantic M [WP] Fantic F [WG] 0.10 0.075 2

3 10 - 14 10 - 14 0.20

0.225 40

42 This product cannot be used in vineyards intended for multiplication or on wine trellises. Argentina Fantic M 2 [WG] 0.10 4 7 - 10 0.40 21 For sensitive varieties, it is a dvised to preventively apply the product pre and post - flowering until the clusters close. To mato Italy Fantic M NC WG; Fantic M blu [WP]

Foliar 0.10 0.10 3

3 7 - 10

10 0.30

0.30 14

14 Begin treatment when there are favorable conditions for the disease or when the main bud has developed (BBCH 61) . Spain Fantic M [WP] 0.10 3 10 0.30 3 Begin application when environmental conditions favor disease development .

Mexico Sidecar M; [WG] Fantic Star [WG] 0.12 0.12 3

3 7

7 0.36

0.36 14

14 Begin application when environmental conditions favor disease development ( t emperatures between 18 and 22 o C and relative humidity above 90%). Do not apply when temperatures are very high, when winds are stronger than 10 km/hour , o r while it is raining. 1 RTI = Retreatment interval. PHI = Preharvest interval. 2 Fantic M use in/on grape is pending in Argentina. The use directions are adequate to allow evaluation of the residue data relative to the proposed uses. The cGAP for grape and tomato were identified as the GAPs from Argentina and Spain, respectivel y.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 10 of 52 **3.4 Anticipated Exposure Pathways**  The Registration Division requested that HED perform an assessment of human health risk to support the petitioner’s request for benalaxyl - M tolerances on imported grapes and tomatoes . No previous Agency

risk assessments have been conducted for benalaxyl - M since there are currentl y no U.S. registrations for benalax yl - M - based products. Humans may be exposed to benalax yl - M through ingestion of imported grapes , tomatoes, and their processed commodities ( i.e. , grape juice , raisins, wine , dried tomato, tomato p aste, puree, etc. ) . Dietary exposures are expected based on detectable residues observed . There are no U.S. drinking water, occupational or residential exposures associated with the requested tolerance . **3.5 Consideration of Environmental Justice**  Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to Address Environmental Justice in Minority Populations and L o w - Income Populations," (http://epa.gov/compliance/ej/resources/policy /exec_order_12898.pdf

) . As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups according to well - established procedures. In line with OPP policy , HED estimates risks to population subgroups from pesticide exposures that are based on patterns of that subgroup’s food and water consumption, and activities in and around the home that involve pesticide use in a residential setting. Extensive data on food consumption patterns are compiled by the USDA under the National Health and Nutrition Survey /What We Eat in America (NHANES/WWEIA) and are used in pesticide risk assessments for all registered food uses of a pesticide. These data are anal yz ed and categorized by subgroups based on age and ethnic group. Additionally , OPP is able to assess die tary

exposure to smaller, specialized subgroups and exposure assessments are performed when conditions or circumstances warrant. Whenever appropriate, non - dietary exposures based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or playing on treated areas postapplication are evaluated. Further considerations are currentl y in development as OPP has committed resources and expertise to the development of specialized software and model s that consider exposure to by standers and farm workers as well as lifestyle and traditional dietary patterns among specific subgroups. **4.0 Hazard Characterization and Dose**  **- Response Assessment**  **4 .1**  **Toxicology Studies Available for Analysis** The database o f experimental toxicology studies available for benalax yl - M provides a robust characterization of the hazard potential of this pesticide. HED has determined that a comparative thy roid assay

in rats is required to assess the potential impact of benalax yl - M exposure on thy roid function in the young (Dellarco et al. , 2005). The agency is requiring these data given the pivotal role of thyroid hormones in brain development. Inthe absence of these data, the 10X FQPA database uncertainty factor has been retain ed. The data from the following studies were used to evaluate the hazard potential of benalaxyl - M:Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 11 of 52  subchronic oral toxicity studies in rats , mice, and dogs  chronic oral toxicity

studies in rats and dogs  carcinogenicity

studies in rats and mice  developmental toxicity studies in rats and rabbits  reproduction toxicity study in rats  acute and subchronic neurotoxicity studies in rats  subchronic dermal toxicity study in rats  immunotoxicity study in rats  complete mutagenicity study battery  metabolism study

in rats **4.2 Absorption, Distribution, Metabolism and Elimination (ADME)**  Benalax yl - M was well absorbed and rapidly excreted. Absorption was rapid and the peak plasma concentrations (T max) were reached within 2 - 6 hours and the terminal half - lives T 1⁄2 ranged from 59 to 102 hrs. There were no major sex differences in the maximum concentration (C max) or in the time taken to reach C max (T max), and the e stimated total absorption was similar between the sexes (~89% for males and ~82% for females). Radiolabel was excreted mainly via the feces within 72 hours (84% of administered dose). Onl y 7% of the administered dose (AD) was excreted via the urine , and t issue radioactivity levels were low. The highest tissue concentrations (as percent of administered dose) 0.5 hours after treatment were found in the liver and intestinal tract , which is i ndicative of active absorption. Total radioactive dose recovered in the tissues 0.5 hours after treatment was 23%. T issue concentrations as percent of administered dose were <1% for both sexes by 72 hours post dose . Metabolism of benalaxyl - M was extensive as at least 18 compounds were found . Benalax yl - M was a minor component excreted in the feces, and it w as not found in the urine. Feces contained a higher number of degradation products , but the main metabolites were identical both in urine and feces. The metabolic profile was the same in male and female rats . Benalax yl - M was extensively metabolized pri ncipally

by oxidation of the methyl group on the aniline ring to the hydroxy methyl and finally

to the carbox yl ic acid. Another metabolic pathway involved hydroxylation of the phenyl ring and hydrol ys is of the carbox ymethyl group. Following single low and high doses, the main route of elimination was fecal via the bile (62 - 81% AD), while urinary excretion (8 - 19% AD) played a minor role. There was evidence of extensive enterohepatic recirculation. Bile was an important route of excretion; a substantial portion of the administered dose was re - absorbed; biliary excretion was substantial for both sexes (80.5% of dose male; 62% of dose females), indicating most of the radiolabeled test material was absorbed and then excreted via the bile into the feces. Excreti on in the expired air was negligible (<0.1%). Residual radioactivity was < 1% AD in the carcasses 96 hours after treatment. **4.2.1 Dermal Absorption**  Dermal exposure is not expected in the U.S. with an import tolerance. There is no dermal absorption study

on benalaxyl - M, but there is a subchronic dermal toxicity study available, which shows no dermal hazard at dose levels up to the limit dose .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 12 of 52 **4.3 Toxicological Effects**  Benalax yl - M is a new fungicide and a member of the N - acylalanine fungicides, which act b y inhibiting RNA poly

merase . There are no data on whether this pesticidal mode of action is relevant to mammalian toxicology . The liver and th yroid are the primary target organs for benalaxyl - M . In rats, increased liver weights, clinical chemi stry changes indicative of liver toxicity, hepatocellular hy pertrophy , and thy roid follicular cell hypertrophy were seen following subchronic and chronic exposure. In mice, increased liver weight and microscopic lesions in the liver (hepatocellular hy pertroph y, necrosis, eosinophilic foci) were observed following subchronic and chronic exposure . Additionally , chronic exposure in rats and mice led to increases in the incidence of liver (rat, mouse) and th yroid (rat) tumors. Indogs, increased liver weight, changes in clinical chemistry indicative of liver toxicity, and hepatocellular hyp ertroph y were observed following subchronic exposure via the diet , whereas clinical chemistry changes indicative of liver toxicity, fat vacuolation in the liver, and thy roid follicular cell hy

pertrophy were observed following chronic exposure via capsules. Benalax yl - M was classified as “Likel y to be Carcinogenic to Humans”. This determination was based on the treatment - related liver tumors observed in male mice, liver tumors observed in male and female rats; and th yr oid follicular cell tumors observed in female rats . No treatment - related tumors were observed in female mice. There is no mutagenicity concern from the in vivo or in vitro genetic toxicity assay s. The negative data rule out mutagenicity as a possible mode of action (MOA) for the induction of tumors in the rat and mouse carcinogenicity studies. A plausible non - genotoxic mode of action involving mitogenesis was not established for the development of liver tumors in the male mouse bioassay with benalax yl - M. M OA data for the liver tumors in male and female rats and the thyroid tumors in female rats were not provided. No evidence of increased quantitative or qualitative susceptibility was seen followi ng in utero exposure to benalax yl - M with rats or rabbits in the prenatal developmental studies or in young rats in the 2 - generation reproduction study . The 2 - generation rat reproduction study resulted in no effects on fertility , although decreased primordial follicles/growing follicles/corpora lutea were observed at the high dose. However, there is a clear NOAEL for the se findings, and there is no reproductive concern. The offspring effects (decreased F 2 pup body weight and increased incidence of hepatoc ellular h ypertroph y in both sexes of F2 pups) occurred at the same dose that caused parental effects (increased liver weight and liver hy pertrophy). No evidence of maternal toxicity and no developmental effects w ere observed in developmental toxicity studies in rabbits or rats. The rabbit was tested at the limit dose (1000 mg/kg/day), but the rat was not tested up to the limit dose . Since the point of departure is 10 - fold lower than the NOAELin the rat study , a repeat rat study

at the limit dose would n ot impact the risk assessment and is not needed. No neurotoxic effects were seen in the acute or the subchronic neurotoxicity study . Benalax yl - M was not found to be immunotoxic in the male rat .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 13 of 52 **4.4 FQ**  **PA Safety F**  **actor for Infants and Children** No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to benalax yl - M with rats or rabbits in the prenatal developmental studies or in young rats in the 2 - generation reproduction study . There is no evidenc e for neurotoxicity following oral exposures to benalaxyl - M . Th yroid toxicity was seen following subchronic and chronic exposures to adult rats. There are , however, no data regarding the potential effects of benalaxyl M on thy roid homeostasis in the youn g animals. This lack of characterization creates uncertainty with regards to potential life stage sensitivities due to exposure to benalax yl - M, and raises the Agency ’s level of concern. Therefore, the Agency is requiring a comparative thy roid assay in rat s with benalax yl - M. This study will better address the concern for potential thy roid toxicity

in the young. There are no residual uncertainties in the benalax yl - M residue database with regards to dietary exposure. T he FQPA Safety Factor is retained at 1 0X in the form of a database uncertainty factor (UF DB ). **4.4.1 Completeness of the Toxicology Database** There are no outstanding studies according to Part 158 data requirements for the current request for an import tolerance on grapes and tomatoes; however, HED has determined that a comparative th yroid assay in rats is required , as discussed above. The uncertainty for the lack of this study is addressed by retaining the FQPA Safety Factor of 10X as the UF DB. **4.4.2 Evidence of Neurotoxicity** There is no evidence for neurotoxicity following oral exposures to benalaxyl - M. Following a single exposure, no evidence for neurotoxicity was observed at the limit dose and twice the limit dose. Following repeated (dietary) exposures, there were no treatment - related clinical signs of neurotoxicity , behavioral changes , or neuropathology . **4.4.3 Evidenc e of Sensitivity/Susceptibility**  **in the Developing or Young**  **Animal** No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to benalax yl - M with rats or rabbits in the prenatal developmental toxicity studies or in yo ung rats in the 2 - generation reproduction study. The 2 - generation reproduction study resulted in no effects on reproductive function or fertility . The offspring effects occurred at the same dose that caused parental effects . No e vidence of developmental delay or developmental toxicity was observed in d evelopmental toxicity studies in rabbits or in rats. The rabbit was tested at the limit dose (1000 mg/kg/day), but the rat was not tested up to the limit dose. Since the point of departure is 10 - fold lower than the NOAEL in the rat study , a repeat rat study at the limit dose would not impact the risk assessment and is not needed. **4.4.4 Residual Uncertainty in the Exposure Database**  There are no residential uses of benalax yl - M in the U.S. and adequate food residue data are available for human health risk assessment. Residue values used in the dietary risk assessments are unlikel y to underestimate risk.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 14 of 52 Dietary exposure assessments were conducted using anticipated residues and assumed 100% crop treated. Therefore, the chronic dietary ( food only) exp osure is considered a conservative estimate . Drinking water exposure in the U.S. is not expected since there are no domestic registrations for benalaxyl - M . **4.5 Toxicity Endpoints and Points of Departure Selections**  **4.5.1 Dose**  **- Response Assessment**  The details for the toxicity endpoint selection are presented in Appendix A2. In risk assessments for import commodities , endpoints are typicall y selected for dietary exposure only. Endpoints for incidental oral, dermal and inhalation exposures are not s elected for import tolerances due to lack of potential U.S. occupational or residential exposure. An acute dietary

endpoint for the general population, including infants and children and females 13+ was not selected for benalax yl - M , based on the lack of toxicity resulting from a single exposure in the database. For chronic dietary

risk assessment, a NOAEL of 20 mg/kg/day was selected based on an increase in gamma glutamyltrans fera se ( GGT ) in males, increased cholesterol in both sexes, increased tri gl yc erides in males, slight increases liver weight in both sexes, increased incidence of hepatocellular h ypertrophy in both sexes, increased incidence of thy roid follicular cell hy pertrophy in both sexes, increased incidence of thy roid cell hy

perplasia in females, increased incidence of th yr oid follicular ectasia in females, and an increased incidence of ovarian stromal cell hy perplasia in females at a LOAEL of 135 mg/kg/day in the chronic toxicity

/ carcinogenicity study in rats . For the POD for the chroni c dietary risk assessment, a total UF of 1000X was applied (10X f

or interspecies variability, 10X for intraspecies vari ability , and 10X for database uncertainty) to account for the lack of a comparative thy roid assay . When the POD is based on thy roid effects, the interspecies extrapolati on UF is ty picall y reduced to 3X due to the pharmacodyn amic differences between rats and humans pertaining to thy roid perturbations. However, in the case of benalax yl - M , the full 10X factor was retained for interspecie s UF since effects not related to thy roid function disruption ( i.e. , liver, ov arian stromal cell hy perplasia) were also the basis of the POD. **4.5.2 Recommendation for Combining Routes of Exposure for Risk Assessment**  Since only

oral exposure is anticipated for benalaxyl - M, it is not relevant to this action to discuss combining routes of exposure at this time. **4.5.3 Cancer Classification and Risk Assessment Recommendations**  In accordance with the EPA’s Final Guidel ines for Carcinogenic Risk Assessment (March, 2005) : the Cancer Assessment Review Committee ( CARC ) classified benalax yl - M as **"Likely to be Carcinogenic to Humans”**  **,** based on the following weight of evidence considerations:Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 15 of 52 (i) Treatment - related hepatocellular adenomas and hepatocellular adenomas and/or carcinomas combined in male rats ; (ii) Treatment - related hepatocellular adenomas in female rats; (iii) Treatment - related thy roid follicular cell adenomas and adenomas and/or carcinomas in female rats; (iv) Treatment - related hepatocellular adenomas and hepatocellular adenomas and/or carcinomas combined in male mice ; (v) Lack of mutagenic concern. Benalax yl - M does not act through a mutagenic mode of action; and (vi) The overall weight - of - evidence was not sufficient to indicate that be nalax yl - M induces liver tumors via a mitogenic m ode of action . The CARC recommended that a low dose extrapolation model (Q 1 *) be applied to the experimental animal tumor data for quantification of cancer risk in human s from exposure to benalax yl - M. The most potent unit risk, Q 1 * , is that for male mouse liver tumor rates at 5.90 x 10 - 3 (mg/kg/day) - 1 in human equivalents. The available data are inadequate to establish a mode of action (MOA) for the male mouse liver tumors. **4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk**  **Assessment Table**  **4.5.4. Summary of Toxicol**  **ogical Doses and Endpoints for B enalaxyl**  **- M for Use in Dietary Hum**  **an Health Risk Assessments**  **Exposure/ Scenario Point of** 

**Departure Uncertainty/** 

**FQPA Safety**  **Fact ors**  **RfD, PAD,**

**Level of Concern for**  **Risk Assessment**  **Study and Toxicological Effects** Ac ute Dietary (General Population, including Infants , Children , and females 13+ ) No appropriate acute endpoint was identified.

Chronic Dietary (All Populations) NOAEL=

20 mg/kg/day UF A= 10x

UF H = 10x FQPA UF DB = 10x Chronic RfD =

cPAD = 0.02 mg/kg/day Chronic Toxicity/Carcinogenicity Study - rat

(49040634) LOAEL = 13 5 mg/kg/day based on based on an increase in γ - glutamyl transferase ( GGT ) in males, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, i ncreased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females. Cancer (oral) Classification: “Likely to be Carcinogenic to Humans” . B ased on male mouse liver tumors ,

Q 1 *= 5.90 × 10 - 3 (mg/kg/day) - 1 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. NOAELBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 16 of 52 = no ob served adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF DB = to account for the absence of a comparative thyroid study. FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (c = chronic). RfD = reference dose. **5.0 Dietary Exposure and Risk**  **A ssessment**  **5.1 Metabolite/Degradate Residue Profile**  **5.1.1 Summary of Plant and Animal Metabolism Studies**  T h e nature of the residue in grape , lettuce, and tomato is adequatel y understood. The parent compound comprised 26 - 44 % of the total residues found in the submitted grape , tomato, and lettuce metabolism studies . The only metabolites found at more than 10 % of the total residues were benzyl alcohol glucoside and malonylg lucoside conjugates of the parent molecule, present at summed levels comparable to the parent molecule . There are no signifi cant livestock feedstuffs associated with grape or tomato . Therefore, no livestock metabolism data are relevant to the present tolerance petition. **5.1.2 Comparison of Metabolic Pathways**  The proposed metabolic pathway s for benalaxyl - M in grape and rat s are provided in Appendix B. Metabolism in lettuce, grape, and tomato shared a common metabolic pathway with parent benalax yl - M being the common and dominant residue. Other major metabolites (>10% TRR) were identified as glucoside or malonylg lucoside con jugates of the parent compound after oxidation. The primary

difference noted in the plant metabolism studies was the extent of benalax yl - M metabolism, which corresponded with the PHI s of the metabolism studies. Thus, relativel y more parent was found in the short - PHI studies (lettuce and tomato) and relativel y less in the longer PHI study

(grape). In the rat metabolism study , benalaxyl - M was extensively metabolized to at least 18 compounds with parent present at only low levels. The primary metabolic p athway involved oxidation of the methyl group on the aniline ring to the hydrox ym ethyl and then to the carboxylic acid. Another pathway involved hydroxylation of the phenyl ring and hydrolysis of the carboxy methyl group. Low levels of glucuronic acid con jugates were identified. Metabolism of benalax yl - M is more extensive in the rat than in plants. The metabolic pathway observed in plants also occurs in the rat , along with other pathway s not found in plants. **5.1.3 Residues of Concern**  **Summary and Rationale**  M ajor residue in the plant metabolism studies were the parent compound and its glucoside and malonylg lucoside conjugates. These conjugates are expected to be stable in the gastrointestinal tract. Conjugation of the parent molecule is likel y to decrease absorption and increase elimination, thereby

reducing the toxicological concern of these residues. Therefore, the residue of concern in plants is parent benalax yl - M for tolerance enforcement and risk assessment.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 1 7 of 52 Given that the only

pathway for exposure at this time is via imported grapes and tomatoes , residue definitions for rotational crops, livestock, and water are not needed. Table 5.1.3 summarizes the residues of concern for benalax yl - M. **Table 5.1.3. Summary of Metabolites and Degradates to be included in th**  **e Risk Assessment and**  **Tolerance Expression** Matrix Residues Included In Risk Assessment Residues Included In Tolerance Expression Plants Primary Crop Benalaxyl - M Benalaxyl - M

Rotational Crop Not Applicable Not Applicable Livestock Ruminant

Poultry Drinking Water **5.2 Food Residue Profile**  The submitted residue chemistry studies are adequate for supporting regulatory conclusions, establishing appropriate tolerances levels for enforcement, and for purposes of risk assessment. Analy sis of residues can be accomplished through standard analy tical techniques, and residues do not dissipate during frozen storage. Following foliar spray applications to grape and tomato, benalax yl - M residues translocate through the plant . Processing studies demonstrate no residue concentration in any

grape or tomato processed commodities except f or raisin, where an average concentration factor of 2.3 × was determined. Multiplying the concentration factor of 2.3× by the grape highest average field trial ( HAFT ) value of 0.98 ppm gives 2.3 ppm, which is less than the re commended grape tolerance of 3 . 0 ppm. Thus, there is no need for a separate raisin tolerance since residue concentration in this processed commodity will be covered by

the grape raw agricultural commodity

( RAC ) tolerance. No confined rotational crop, livestock metabolism, or livestock feeding studies were submitted or required. At this time there are no U.S . registrations for benalax yl - M. E stablishment of U.S. tolerances for grape and tomato was requested to support the import of treated crops from Europe, Central America, and South America. For grape and tomato the maximum application rates are similar across countries; thus, critical Good Agriculture Practice (cGAP) selection was based on the shortest PHI , since residue decline trials showed a gradual decrease in residues with in creasing PHI . The Argentina use pattern constitutes the c GAP for grape, and the recommended grape tolerance of 0.20 ppm was based on trials reflecting the cGAP. Three trials from Argentina conducted in 2011 gave residues approximatel y ten times higher th an residues reported in any of the other grape trials and prevent harmonization with the Codex MRL for benalax yl . For tomato, the Spain use pattern, with a preharvest interval ( PHI ) of 3 days, was identified as the cGAP and formed the basis for the recomm ended tomato tolerance, which is harmonized with the Codex MRLfor benalax yl .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 18 of 52 **5.3 Water Residue Profile**  An assessment of residues in drinking water is not required for this assessment because there is no drinking water exposure in the U.S. associated with the establishment of an import tolerance. **5.4 Dietary Risk Assessment**  **5.4.1 Description of Residue Data Used in Dietary Assessment**  For the chronic and cancer dietary anal ys e s, anticipated residues ( i.e., mean field trial residues) were used. The r esidue chemistry database for benalaxyl - M included processing studies for all relevant processed commodities from grape and tomato except for dried tomato. Thus, the default processing factor of 14.3× was used for dried tomato. The only processed commodi ty showing residue concentration was for raisin, with an average concentration factor of 2.3×. All other processed commodities showed residue reduction upon processing; for these commodities a processing factor of 1.0 was used in the analy sis. The Agency considers these to be conservative assessments. **5.4.2 Percent Crop Treated Used in Dietary Assessment**  For the chronic and cancer dietary anal ys e s, a 100% crop treated assumption was used **. 5.4.3**  **Acute Dietary Risk Assessment** Because no acute endpoint was identified in the toxi city database, no acute dietary anal ys is was conducted. **5.4.4 Chronic Dietary Risk Assessment**  The results of the chronic dietary exposure anal ys es are reported in Table 5.4.6. The resulting chronic risk estimates were less than HED’s level of concern ( ≤ 100% cPAD) for the general U.S. population ( 1.4 % cPAD ) and all population sub - groups. Th e most highl y exposed population subgroup was Children 1 - 2 ye ar s old with an estimated risk of 7.1 % cPAD. **5.4.5 Cancer Dietary Risk Assessm**  **ent** The results of the cancer dietary exposure anal ys es are reported in Table 5.4.6. The cancer dietary assessment made use of the same input assumptions as the chronic analy sis. Benalax yl M has been classified as “Likely to be Carcinogenic to Humans” (TXR No. 0057093, L . Brunsman, 04 - DEC - 2014). A linear low - dose extrapolation model (Q 1 *) was used to estimate cancer risk, with a Q 1 * = 5.90 × 10 - 3 (mg/kg/day) - 1 . The cancer risk estimate to the U.S. population is 1.7 x 10 - 6

.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 19 of 52 **5.4.6 Summary Table**  **Table 5.4.6 . Summary of Dietary**  **(Food Only** **) Exposure and Risk for**  **Benalaxyl -**  **M. 1**  Population Subgroup Ac ute Dietary Chronic Dietary Cancer Dietary Exposure (mg/kg/day) % aPAD Dietary Exposure (mg/kg/day) % cPAD Dietary Exposure (mg/kg/day) Risk **General U.S. Population** 

NA NA 0.000287 1.4 **0.000 287**  **1.7 ×**  **10 -**  **6** All Infants (< 1 year old) 0.000477 2.4

NA NA **Children 1 -**  **2 years old 0.001416**  **7.1** Children 3 - 5 years old 0.000901 4.5 Children 6 - 12 years old 0.000381 1.9 Youth 13 - 19 years old 0.000148 < 1 Adults 20 - 49 years old 0.000201 1.0 Adults 50 - 99 years old 0.000225 1.1 Females 13 - 49 years old 0.000206 1.0 1 Highest exposure identified in bold. **6.0 Residential (Non**  **- Occupational) Exposure/Risk Characterization**  There are no registered residential uses for benalaxyl - M . **7.0 Aggregate Exposure/Risk Characterization**  As stated previousl y, there are no existing or proposed US registrations of benalax yl - M and the only route of exposure is via dietary ingestion from imported grape and tomato commodities. Therefore, aggregate exposure and risk estimates are equivalent to the dietary exposures and risk estimates discussed in Section 5. **8.0 Cumulative Exposure/Risk Characterization**  Unlike other pesticides for which EPA h as followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to benalax yl - M and any other substances and benalax yl - M does not appear to produce a toxic metabolite produced b y oth er substances. For the purposes of this tolerance action, there fore, EPA has not assumed that benalax yl - M has a common mechanism of toxicity with other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy statements released by

EPA’s Office of Pesticide Programs concerning common mechanism determinations and procedures for cumulating effects from substances found to haveBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 20 of 52 a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/. **9.0 Occupational Exposure/Risk Characterization**  An occupational assessment is not relevant for uses on imported commodities . **10 .0**  **References** Dellarco, V. et al. 11/01/2005. Interim Guidance: Thy roid Disrupting Pesticides: Use of Rat Thy roid Data and Application of Uncertainty Factors for RfD Derivation. Prepared and Reviewed b y the Hazard Science Policy Council. OPP Guidance. 10/24/2005. Guidance for Th yroid Assays in Pregnant Animals, Fetuses and Postnatal Animals, and Adult Animals. Office of Pesticide Programs, Health Effects Division, Washington, DC. Cited in Dellarco, V. et al 2005 Benalax yl - M . Report of th e Residues of Concern Knowledgebase Subcommittee (ROCKS).

– E. Holman , D 421423 , 09/09 /1 4 . Benalax yl - M. Report of the Cancer Assessment Review Committee. J. Rowland and K. Middleton, TXR 0057035, 12/02/14. Benalax yl - M. Quantitative Risk Assessment Based on Rat and Mouse Dietary Studies. L . Brunsman, TXR 0057093, 12/04/14. Residue Chemistry

Assessment

– W. Donovan , D416583 , 1 2 / 02/14 . Dietary Exposure Assessment

– W. Donovan , D 416584 , 01 / 1 3 /15 .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 21 of 52 **Appendix A. Toxicology Profile and Executive Summaries**  **A.1 Toxicology Data Requirements**  The requirements (40 CFR 158.340) for food (import tolerance ) for benalaxyl - M are in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used. **Study Technical**  **Required Satisfied**  870.1100 Acute Oral Toxicity ................................ ....................... 870.1200 Acute Dermal Toxicity ................................ .................. 870.1300 Acute Inhalation Toxicity ................................ .............. 870.2400 Primary Eye Irritation ................................ .................... 870.2500 Primary Dermal Irritation ................................ .............. 870.2600 Dermal Sensitization ................................ ...................... y es no no no no no y es

---

---

---

---

--- 870.3100 Oral Subchronic (rodent) ................................ ............... 870.3150 Oral Subchronic (nonrodent) ................................ ......... 870.3200 21 - Day

Dermal ................................ .............................. 870.3250 90 - Day

Dermal ................................ .............................. 870.3465 90 - Day

Inhalation ................................ .......................... y es no no no no yes

yes

---

---

--- 870.3700a Developmental Toxicity (rodent) ................................ ... 870.3700b Developmental Toxicity (nonrodent) ............................. 870.3800 Reproduction ................................ ................................ . y es yes yes yes

yes yes 870.4100a Chronic Toxicity (rodent) ................................ .............. 870.4100b Chronic Toxicity (nonrodent) ................................ ........ 870.4200a Oncogenicity (rat) ................................ .......................... 870.4200b Oncogenicity (mouse) ................................ .................... 870.4300 Chronic/Oncogenicity ................................ .................... y es yes yes yes yes yes

yes yes yes yes 870.5100 Mutagenicity

—Gene Mutation - bacterial ..................... 870.5300 Mutagenicity

—Gene Mutation - mammalian ................ 870.5 375 Mutagenicity

— Structural Chromosomal Aberrations ... 870.5xxx Mutagenicity

—Other Genotoxic Effects ....................... yes yes yes no yes

yes yes

--- 870.6100a Acute Delayed Neurotoxicity (hen) ............................... 870.6100b 90 - Day

Neurotoxicity (hen) ................................ ........... 870.6200a Acute Neurotoxicity Screening Battery (rat) ................. 870.6200b 90 - Day

Neurotoxicity Screening Battery (rat) ............... 870.6300 Develop. Neurotoxicity ................................ ................. no no yes yes no ---

--- yes yes

--- 870.7485 General Metabolism ................................ ...................... 870.7600 Dermal Penetration ................................ ........................ 870.7800 Immunotoxicity ................................ ............................. yes no yes yes

--- y esBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 22 of 52 **A.2 T**  **oxicity Profiles A.2.1**  **Acute Toxicity**  **Table A.2.1 Acute Toxicity Profile**  **– Benalaxyl**  **- M**  **(technical) Guideline No.**  **Study Type MRID(s)**  **Results Toxicity Category**  870.1100 Ac ute oral [rat] 49040619 LD 50 = >2000 mg/kg No deaths or clinical signs III 870.1200 Ac ute dermal [rat] 49040620 LD 50 = >2000 mg/kg III 870.1300 Ac ute inhalation [rat] 49040621 49040622 LC 50 = mg/L

Aerosol could not be generated (waiver granted) - 870.2400 Ac ute eye irritation [rabbit] 49040623 No corneal opacity or iritis; + score of 2 for conjunctival redness at 24 hrs; no + scores at 48 hrs; all eyes free of irritation at 72 hrs III

870.2500 Ac ute dermal irritation [rabbit] 49040624 Not an irritant IV 870.2600 Skin sensitization [Guinea pig] 49040625 Not a sensitizer - **A.2.2 Subchronic, Chronic, Other Toxicity**  **Table A.2.2 Subchronic, Chronic and Other Toxicity Profile**  **– Benalaxyl**  **- M**  **Guideline No./ Study Type**  **MRID No. (year)/**

**Classification /Doses Results**  870.3100 90 - Day Oral Toxicity (Crl:CD BR rat) 49040627 (2000) Acceptable/guideline 0, 80, 400, 2000, or 10000 ppm (diet) Males: 0, 6.2, 30.1, 154, or 861 mg/kg/day Females: 0, 6.7, 32.8, 167, or 892 mg/kg/day N OAEL = 2000 ppm (males 154 mg/kg/day; females 167 mg/kg/day). LOAEL = 10000 ppm (males 861/females 892 mg/kg/day), based on elevated GGT values in both sexes, increase d cholesterol and phospholipids in both sexes, increased liver weights in both sexes, incre ased thymus weight in females, increased incidence of hepatocellular hypertrophy in both sexes , and increased incidence of thyroid follicular cell hypertrophy in both sexes. F indings at 2000 ppm are considered to be minor [increased GGT (both sexes); increased phospholipids (males), a daptive hepatocellular hypertrophy (both sexes), and not biologically adverse due to lack of severity (thyroid follicular cell hypertrophy]Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 23 of 52 **Table A.2.2 Subchronic, Chronic and Other Toxicity Profile**  **– Benalaxyl**  **- M**  **Guideline No./ Study Type**  **MRID No. (year)/**

**Classification /Doses Results**  870.3100 90 - Day Oral Toxicity (CD - 1 mouse) 49040628 (2005) Acceptable/guideline 0, 600, 2000, or 7000 ppm (diet) Males: 0, 93.2, 296.6, or 1053.8 mg/kg/day Females: 0, 111.8, 348.1, or 1253.3 mg/kg/day Sy stemic NOAEL = 2000 ppm (296.6 mg/kg/day in males and 348.1 mg/kg/day in females). Sy stemic LOAEL = 7000 ppm (1054 mg/kg/day in males and 1253 mg/kg/day in female s), based on increased liver weights, increased incidence and severity of periportal hepatocellular hypertrophy in females, increased glucose in the urine in both sexes, focal necrosis in the liver in males, and depleted glycogen in the liver in females. 870.3150 90 - Day Oral Toxicity (Beagle dog) 49040629 (2000) Acceptable/guideline 0, 150, 450, 900, or 3000 ppm ( **diet** ) Males: 0, 5.6 , 16 , 32 , or 112 mg/kg bw/day Females: 0, 5.85 , 17 , 35 , or 110 mg/kg bw/day Sy stemic NOAEL = 90 0 ppm (32 mg/kg/day in males and 35 mg/kg/day in females). Sy stemic LOAEL = 30 00 ppm ( 112 mg/kg/day in males and 110 mg/kg/day in females), based on elevated alkaline phosphatase activity in both sexes, elevated triglyceride levels in males, increased liv er weight in both sexes, and an increased incidence/ severity of hepatocyte hypertrophy in the centrilobular, midzonal, and periportal areas in males and females. 870.3200 28 - Day Dermal Toxicity (Wistar rat) 49040631 (2013) Acceptable/guideline 0, 62.5, 250, or 1000 mg/kg/day 6 hours/day, 7 days/week for 4 weeks NOAEL = 1000 mg/kg/day LOAEL = not identified Endpoints of concern (liver, thyroid) were assessed; no developmental, neurotoxicity, immunotoxicity, or reproductive concern. 870.3465 28/90 - Day Inhalation Toxicity 49066308 Waiver request 49040621/49040622 See MRID49040621 - 49040622 (acute inhalation) 870.1300 waived. Aerosol could not be generated. 870.3700a Prenatal developmental (Crl: CD (SD) BR rat) 49049637 (2001) A cceptable/non guideline 0, 10, 50, or 250 mg/kg/day (dose volume 10 mL/kg/day) (0.5% methylcellulose aqueous solution) GD 6 - 15 **Maternal** NOAEL = 250 mg/kg/day (HDT) **Maternal** LOAEL = not identified **Developm ental**  NOAEL =250 mg/kg/day **Developm ental**  LOAEL = not identified Non - guideline since it did not test to limit dose and no adverse effect was observed in the maternal animals. A new study is not required since an endpoint more sensitive than the one used for risk assessment would not be identified by a study at dose leve ls >250 mg/kg/day 870.3700b Prenatal developmental in (Himalayan rabbit) 49040638 (2003) A cceptable/guideline 0, 50, 250, or 1000 mg/kg/day (0.5% methylcellulose) GD 6 - 27 (dose volume 4 mL/kg/day) **Materna** l NOAEL = 1000 mg/kg/day (HDT) **Maternal** LOAEL = not identified **Developm enta**  l NOAEL = 1000 mg/kg/day **Developm enta**  l LOAEL = not identifiedBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 24 of 52 **Table A.2.2 Subchronic, Chronic and Other Toxicity Profile**  **– Benalaxyl**  **- M**  **Guideline No./ Study Type**  **MRID No. (year)/**

**Classification /Doses Results**  870.3800 Reproduction and Fertility Effects (Han Wistar rats) 49040639 (2008) A cceptable/guideline 0, 120/80, 360/240, 1080/725, or 3240/2170 ppm Males: 0, 7.9, 23.5, 71.1, or 221.3 mg/kg/day Females 0, 8.9, 26.7, 77.5, or 241.8 mg/kg/day 70 - day pre - mating **Parental** NOAEL = 1080 ppm (males 71/females 78 mg/kg/day) **Parental** LOAEL = 3240 ppm (males 221/females 242 mg/kg/day), based on decreased # primordial fol licles and corpora lutea in F1 females, decreased uterus weight in F0 females , and a slight increase in hepatocellular hypertrophy in both sexes and generations (males P 16/24, F1 12/24; females P 21/24; F1 23/24),. **Offspring** NOAEL = 1080 ppm (males 71/females 78 mg/kg/day) **Offspring** LOAEL = 3240 ppm (males 221/females 242 mg/kg/day), based on decreased pup body weight in F 2 pups of both sexes and increased incidence of hepatocellular hypertrophy in F 2 pups of both sexes. **Reproductive** NOAEL = 1080 p pm (males 71/females 78 mg/kg/day) **Reproductive** LOAEL = 3240 ppm (males 221/ females 242 mg/kg/day), based on a decrease in ovarian follicle counts (primordial follicles and corpora lutea) in F1 females . outside historical control and concurrent control va lues were generally low

compared to the historical control values. 870.4100a Chronic Toxicity (Sprague Dawley CD Crl:CD(SD)BR rat) 49040634 (2013) See under 870.4200a 870.4100b Chronic toxicity (Beagle dog) 49040630 (2006) Acceptable/guideline 0, 10/300 A , 30, or 100 mg/kg/day (52 weeks) **capsule** Alow dose ↑to 300 mkg (week 27) NOAEL = not selected. LOAEL = not selected.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 25 of 52 **Table A.2.2 Subchronic, Chronic and Other Toxicity Profile**  **– Benalaxyl**  **- M**  **Guideline No./ Study Type**  **MRID No. (year)/**

**Classification /Doses Results**  870.4200a Carcinogenicity (Sprague Dawley CD Crl:CD(SD)BR rat) 49040634 (2013) A cceptable/guideline 0, 6 0 , 390 , or 2535 ppm Main study: Males 0, 2.4, 15.66, 104.27 mg/kg/day Females 0, 3.13, 20.04, or 135.28 mg/kg/day 52 - we ek chronic study: Males: 0, 2.92, 19, 126 mg/kg/day Females: 0, 3.82, 24.6, or 167 mg/kg/day NOAEL = 390 ppm ( ma les 15.7 mg/kg/day/females 20 mg/kg/ day . LOAE L = 2535 ppm

( males 104/females 135 mg/kg/day), based on increase in GGT in males, increased cholesterol in both sexes, increased triglycerides in males, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females . **evidence of carcinogenicity:** increase in liver tumors (adenomas) at the high dose in both sexes ; increase in thyroid follicular cell adenomas and follicular cell adenomas/carcinomas combined in high dose females ( CARC 8 /1 3/2014; Likely/Q* 1

). 870.4200b Carcinogenicity (78 weeks) (CD - 1 mouse) 49040635 (2013) Acceptable/guideline 0, 200, 1200, or 7000 ppm Males: 0, 24.86, 156.1, or 962 mg/kg/day Females: 0, 31.36, 196.7, or 1180 mg/kg/day Sy stemic NOAEL = 1200 ppm (156 mg/kg bw/day for males and 197 mg/kg/day for females). Sy stemic LOAEL = 7000 ppm (962 mg/kg bw/day for males and 1180 mg/kg bw/day for females) , based on increased liver weight in both sexes and an increased incidence of liver lesions (diffuse hepatocellular hypertrop hy in both sexes, single cell necrosis in both sexes, focal/ multifocal necrosis in males, and eosinophilic foci in males ). **evidence of carcinogenicity:** increased incidence of liver adenomas in males and liver adenomas/ carcinomas in both sexes at HDT; ( CARC 8 /1 3/2014; Likely/Q* 1 ). 870.4300 Com bined Chronic Toxicity/Carcinogenicity (Sprague Dawley CD Crl:CD(SD)BR rat) 49040634 (2013) See under 870.4200a

870.5100 Bacterial reverse gene mutation (Ames Assay) 49040640 (2002) Acceptable/guideline 5 - 5000 μg/plate +/

– S9 (plate incorporation) 5 - 1500 μg/plate +/

– S9 (preincubation) Negative up to the limit dose; compound precipitation at ≥ 1500 μg/plate

870.5300 In vitro mammalian forward cell gene mutation (mouse lymphoma cells) 49040642 (1999) Acceptabl e/guideline 5 - 75 μg/mL

– S9 15 - 100 μg/mL +S9 Negative up to cytotoxic concentrationBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 26 of 52 **Table A.2.2 Subchronic, Chronic and Other Toxicity Profile**  **– Benalaxyl**  **- M**  **Guideline No./ Study Type**  **MRID No. (year)/**

**Classification /Doses Results**  870.5375 In vitro mammalian chromosome aberration test [Chinese Hamster Ovary (CHO cells)] 49040641 (1999) Acceptable/guideline Assay 1 5 - 3000 μg/mL+/

– S9 (3 hrs.) **Assay 2** 5 - 150 μg/mL

– S9 (18 hrs.) 15 - 150 μg/mL +S9 (3 hrs.) Negative up to cytotoxic & insoluble concentration

870.5395 Mammalian erythrocyte micronucleus assay (mouse) 49040643 (2000) Acceptable/guideline 0, 100, 200, 400 mg/kg M 0, 75, 150, 300 mg/kg F Single i.p. injections Negative up to doses ( ≥300 mg/kg) causing overt toxicity in preliminary study (deaths and/or piloerection) 870.6200a Ac ute Neurotoxicity Screening Battery (CRL Wistar rat) 49040626 (2013) Acceptable/guideline 0, 500, 1000, or 2000 mg/kg 10 mL/kg dose volume 0.5% aqueous CMC (peak effect time 30 minutes) NOAEL = 2000 mg/kg LOAEL = not identified.

870.6200b Subchronic Neurotoxicity Screening Battery (CRL Wistar rat) 49143501 (2013) Acceptable/guideline 0, 1000, 3000, or 10000 ppm Males: 0, 67, 206, or 784 mg/kg/day Females: 0, 78, 325, or 1139 mg/kg/day Sy stemic NOAEL = 3000 ppm (males 206 /females 325 mg/kg/day . Sy stemic LOAEL = 10000 ppm ( males 784/females 1139 mg/kg/day , based on increased serum GGT activity in both sexes and increased absolute and relative liver weights. No neurobehavioral or neuropathological effects were observed in either sex. 870.6300 Developmental Neurotoxicity No study available or required. 870.7485 Metabolism and Pharmacokinetics (Sprague - Dawley rat) 49040644 A cceptable/guideline Radiolabel readily absorbed and excreted mainly via the feces ( ≈85%) and to a smaller extent via urine ( ≈8%). Biliary excretion explains high elimination of absorbed radiol abel via fecal route. By 72 hours, all tissue concentration were <1%; highest tissue concentrations in intestinal wall (8%), liver (8%), and stomach (6%); PK profile characterized by a peak level between 10 minutes and 1 hour post dose; elimination half - li fe of ≈18 hours; IR6141 was extensively metabolized, principally by oxidation of methyl group on aniline ring to the hydroxymethyl and finally to the carboxylic acid; minor pathways were hydroxylation of phenyl ring and hydrolysis of carboxymethyl group. s hows urinary metabolic pathway identical to fecal metabolic pathway. 870.7485 Metabolism and Pharmacokinetics (Sprague - Dawley rat) 49040645 (1999) A cceptable/guidelineBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 27 of 52 **Table A.2.2 Subchronic, Chronic and Other Toxicity Profile**  **– Benalaxyl**  **- M**  **Guideline No./ Study Type**  **MRID No. (year)/**

**Classification /Doses Results**  870.7485 Metabolism and Pharmacokinetics (Sprague - Dawley rat) 4904046 ( 2008) A cceptable/guideline Total absorption similar to that in studies above, although females excreted more in urine than males (Females 17% vs males 8%); however, biliary excretion was substantial in both sexes (males 81%; females 62%), indicating most of radi olabel was absorbed and then excreted via the bile into feces; estimated total absorption was similar (males 89%; females 82%); recovery 97% - 99%. Highest tissue concentr ations in liver and GI tract in both sexes; T max wa s 1.8 hours post dose; terminal half - life of 62.4 hours; data suggest that absorption and elimination were not saturated since there was not a 10 - fold increase in Cmax and tmax between the 10 mkd and 100 mkd doses. 870.7485 Metabolism and Pharmacokinetics (Sprague - Dawley rat) 49040647 (2008) A cceptable/guideline

870.7600 Dermal penetration No study available 870.7800 Immunotoxicity (Wistar rat, male) 49040648 (2013) A cceptable/guideline 0, 1000, 3000, or 10000 ppm (28 days) 0, 80.5, 250.4, or 924.6 mg/kg/day Sy stemic NOAEL = 1000 ppm (80.5 mg/kg/day) Sy stemic LOAEL = 3000 ppm (250.4 mg/kg/day), based on increased absolute and relative - to - body liver weights. Immunotoxicity NOAEL = 10000 ppm (924.6 mg/kg/day) Immunotoxicity LOAEL = not identified. Non - guideline (mechanistic) (Crl:CD - 1(Icr) BR mouse) 49040636 (2013) A cceptable/non guideline 0, 200, 1200, or 7000 ppm (diet; 3 or 14 day) 0, 30.9, 184.4, or 1094.1 mg/kg/day (males) Assessment of cell proliferation not adequate; microsomal liver enzymes EROD, MROD, and LA12OH activ ities : slightly increased at 1200 and 7000 ppm ; PROD, 6β - OH, and LA11OH activities : mo derately increased at 7000 ppm; PROD activity : mo derately increased at 1200 ppm ; and 6β - OH and LA11OH activities : slightly increased at 1200 ppm. E xpression of the Cyp1A - 1 , Cyp1A - 2 , Cyp2B10 , and Cyp3A11 genes were increased at 7000 ppm ; expression of Cyp1A - 1 , CypB10 , and Cyp3A11 we re increased at 1200 ppm ; expression of Cyp2B10 wa s increased 45.8 - and 195.7 - fold at 1200 and 7000 ppm, respectively ; expression of Cyp3A 11 wa s increased 15.58 - fold. Examination of the parameters of oxidative stress, reduced glutathione (GSH) and oxidized glutathione (GSSH) in liver, and thiobarbituric acid reactive substance (TBARS) in liver and serum, showed no effect of treatment with IR6141 for 14 days.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 28 of 52 **A.3 H**  **azard Identification & Endpoint Selection A.3.1**  **Acute Reference Dose (aRfD) –**  **All Populations** An endpoint for an aRfD for either females age 13 - 49 or the general population was not identified in the toxicity

database, including acute neurotoxicity or developmental toxicity studies. **A.3.2. Chronic Reference Dose (cRfD**  ) **Study Selected:** 2 - year carcinogenicity study in rats **MRID No.: Executive Summary**  **:** See Appendix A.4.5, Guideline § 870.4200.a **Dose and Endpoint for Risk Assessment:** NOAEL= 20 mg/kg/day based **Comments about Study/Endpoint/Uncertainty Factors:** This study was the most sensitive endpoint and is the appropriate duration and route of exposure. The LOAEL of 135 mg/kg/day is based on an increase in GGT in males, increased trigl ycerides in males, increased cholesterol in both sexes, slight increases liver weight in both sexes, increa sed incidence of hepatocellular hy pertrophy in both sexes, increased incidence of thy roid follicular cell hypertrophy in both sexes, increased incidence of thy roid cell hy

perplasia in females, increased incidence of thyr oid follicular ectasia in females, and an increased incidence of ovarian stromal cell hy perplasia in females . A 10X interspecies uncertainty factor to extrapolate from animals to humans; a 10X uncertainty factor to account for potential variation in sensitivity among the human population; an d a 10X database uncertainty factor to account for the lack of a critical study (comparative thy roid assay (CTA) ) were used. For the POD for the chronic dietary risk assessment, a total uncertainty factor of 1000X was applied (10X for interspecies variabil ity , 10X for i ntraspecies variability , and 10X for database uncertainty to account for the lack of a CTA study). When the basis of the POD is based on thy roid effects, the interspecies extrapolation UF is ty pically reduced to 3 X due to the pharmacodyn amic differences between rats and humans pertaining to thy roid perturbations. However, in the c ase of benalaxyl - M, the full 10X factor was retained for interspecies UF since effects not related to thyroid function disruption are also the basis of the POD. **A.3.3 Incidental Oral Exposure, Dermal Exposure, Inhalation Exposure**  Endpoints for these exposure routes are not needed to assess non - domestic uses . **A.4 E**  **xecutive Summaries A.4.1**  **Subchronic Toxicity 870.3100**  **90 -**  **Day Oral Toxicity -**  **Rat**Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 29 of 52 In a subchronic oral toxicity study (MRID 49040627 ) , five groups of Crl: CD BR rats (10/sex/group; ~ 5 weeks of age) were administered 0, 80, 400, 2000, or 10,000 ppm of benalax yl - M [97.27% (Batch No. FCT/T/156 - 99 (20398/15)] in the diet and observed for 90 day s. The respective dose levels based on food consumption were 0, 6.2 , 30.1 , 154, and 861 mg/kg bw/day

for males and 0, 6.7, 32.8, 167, and 892 mg/kg bw/day for females. The animals in each test group were subjected to neurobehavioral testing, clinical pathology , and gross and microscopic examinations. There was no adverse effect on survival for either sex, and no clinical signs attributed to the test substance were observed at any dietary concentration. Both sexes showed body weight deficits of 12% - 14% throughout the 13 - week exposure period at 10000 ppm, which may be attributed to an initial aversion to the diet. Decreased food consumption was observed in both sexes during the first week of the study

but was comparable to the control thereafter No statistically

significant differences were observed between any treatment group and the controls during the functional operational battery ( FOB ) and locomotor testing. The minor changes in hematology

parameters were not considered adverse. The most notable changes in the clinical chemistry

data were dose - related elevations in GGT activity in both sexes at the two highest dietary

concentrations, elevations in cholesterol at the highest dietary concentration in both sexes, and elevations in ph ospholipids at the mid - high and high concentration in males and at the high concentration in females. There were no treatment - related or toxicologically significant findings in the urinal ys is or gross examination findings. Males and females in the high do se group had statistically higher absolute and relative liver weights compared to controls. Females in the mid - high dose group had statistically higher relative liver weights . Males in the high dose group had statistically higher relative brain, thy roid, t estes, and epididy

mides weights compared to controls , which were likely related to the lower body

weight at this dietary

concentration . Females in the high dose group had statistically higher absolute and relative thy mus weights , and lower absolute and rel ative heart weights. Generalized hepatocy

te hy pertrophy was observed in all males and females in the high dose group and in 8/10 males and 2/10 females in the mid - high dose group. Increased th yroid follicular cell h ypertrophy was observed in five males and three females at the high dose and in three males at the mid - high dose compared to none in the control, low - and low - mid dose groups. There was an increase in the incidence and prominence of vacuolated cells in the pituitary pars distalis in six males at the high dose. There was an increased incidence of kidney cortico medullary

mineralization in females at the high dose (10/10) compared to the control (6/10) and other dose groups (5/10). **The NOAEL was 2000 ppm (males 154 mg/kg/day; females 167 mg/kg/da y), based on**  **elevated GGT values in both sexes, increased cholesterol and phospholipids in both sexes,**  **increased liver weight in both sexes, increased thymus weight in females, increased incidence of hepatocellular hypertrophy (both sexes), and increased t**  **hyroid follicular cell hypertrophy (in both sexes) at the LOAEL of 10000 ppm (males 861 mg/kg/day; females**  **892 mg/kg/day.**Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 30 of 52 This subchronic neurotoxicity study is classified as **Acceptable/Guideline** and satisfies the guideline requirement for a 13 - week toxicity study in rats (OCSPP 870.3100; OECD 408). **870.3100 90**  **- Day Oral Toxicity**  **- Mouse**  In a subchronic oral toxicity study (MRID 49040628 ) , four groups of CD - 1 mice (10/sex/group; 6 weeks of age) were administered 0, 600, 2000, or 7,000 ppm of benalaxyl - M [96.33% (Batch No. G018/04)] in the diet and observed for 90 days. The respective dose levels based on food consumption were 0, 93.2 , 296.6, and 1053.8 mg/kg bw/day for males and 0, 111.8 , 348.1, and 1253.3 mg/kg bw/day

for females. The animals in each test group were subjected to clinical pathology testing, and gross and microscopic examinations. All animals survived to scheduled sacrifice. No treatment - related clinical signs or body wei ght effects were observed. The most notable statistically significant clinical chemistry finding was a test material - related elevation in total bilirubin (p<0.01) in females at the high dietary concentration. Urinal ys is data showed a treatment - related e lev ation in glucose in males and females at the mid and high dose level s. Males and females in the high dose group had statistically

higher absolute and relative liver weights compared to controls (p ≤0.01) . Females in the mid - high dose group had statistically higher relative liver weights (p ≤0.01). Minimal to slight periportal hepatocellular hy pertrophy (without signs of necrosis) was observed in both sexes in all treated dose groups (none in controls), but only the females showed a dose - response . The average grade of hepatocellular gl yc ogen deposits decreased in both sexes in all treated dose groups, but onl y the females showed a dose - response. **The LOAEL in mice was 7000 ppm (1054 mg/kg/day in males and 1253 mg/kg/day in females), based on increased liver we**  **ights, increased incidence and severity of periportal hepatocellular hypertrophy in females, increased glucose in the urine in both sexes, focal**  **necrosis in the liver in males, and depleted glycogen in the liver in females. The NOAEL is 2000 ppm (296.6 mg/**  **kg/day in males and 348.1 mg/kg/day in females).** This subchronic oral toxicity study is classified as **Acceptable/Guideline** and satisfies the guideline requirement for a 13 - week toxicity study in a rodent (OCSPP 870.3100; OECD 408). **870.3150 90**  **- Day Oral**  **Toxicity –**  **Dog** In a 13 - week oral toxicity study (MRID 49040629 ) , five groups of beagle dogs (4/sex/group; 23 - 36 weeks of age) were administered 0, 150, 450, 900, or 3000 ppm of benalaxyl - M [97.27% (Batch No. FCT/T/156 - 99 (20398/15))] via the diet . The respective dose levels based on food consumption were 0, 5.58, 16.24, 32.02, and 112 mg/kg bw/day for males and 0, 5.85, 17.34, 34.97, and 110 mg/kg bw/day for females. The animals in each test group were subjected to clinical pathology , and gross and microscopic examinations. All animals survived to the end of the study. No clinical signs attributed to the test substance were observed at any dietary concentration. Body weights were slightly lower at the 3000 ppm dose level in both sexes at termination (males ↓4%/females ↓8%) compared to the control groups. Food consumption was not adversel y affected in either sex. The most notable changes inBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 31 of 52 clinical chemistry

data were statisticall y elevated alkaline phosphatase activity (ALK) in both sexes (males ↑353% - 4 94%/females ↑287% - 374%) at the high dietary concentration, with the magnitude of the effect increasing with time. ALK was also elevated at the mid - high concentration in males ( ↑110% - 141%) at both the 6 - and 13 - week sampling intervals and in females ( ↑85%) at the mid - high concentration at the 13 - week interval. The historical control range for ALK activity

was exceeded in males and females at the high dietary concentration. Trigl yc erides were significantly elevated at the high dose level in males ( ↑70% - 96%) at 6 and 13 weeks and elevated in high - dose females ( ↑40% - 68%) at the same time points but statistical significance was not attained. There were no treatment - related or toxicologically significant findings in the hematology or urinal ys is data. Enlarged liver was observed in 2/4 males and 1/4 females at the high dietary concentration during gross examinations . Males and females in the high dose group had statistically higher absolute ( ↑19%) and relative (↑23% - 29%) liver weights compared to controls (p ≤0.01) . Hepatocy te hy pertrophy in centrilobular and midzonal areas were observed in all males and females in the mid - high and high dose groups. The change extended to the periportal area in two males and two females at the high dose and in one female from the mid - high dose group. No signs of liver necrosis were observed. **The no -**  **observed -**  **adverse effect level (NOAEL) for benalaxyl**  **- M**  **in beagle dogs was 900 ppm (32 mg/kg/day in males and 35 mg/kg/day in females). A lowest** **- observed**  **- adverse**  **effect level (LOAEL) for IR 6141 in beagle dogs was 3000 ppm (112 mg/kg/day in males and 110 mg/kg/day in females), based on elevated alkaline phosph**  **atase activity in both sexes, elevated triglyceride levels in males, increased liver weight in both sexes, and an**  **increased incidence/severity of hepatocyte hypertrophy in the centrilobular, midzonal, and periportal areas in males and females.**  This subchronic toxicity

study in the dog is classified as **Acceptable/Guideline** and satisfies the guideline requirement for a 13 - week toxicity study in non - rodents (OCSPP 870.3150; OECD 409). **870.3200 21/28**  **- Day Dermal Toxicity**  **– Rat**  In a 28 - day

dermal toxicity study

(MRID 49040631 ), benalax yl - M t echnical ( 96.53% a.i., Lot No. P/10/106 ) was applied to the shaved skin of 10 Wistar rats/sex/group at concentrations of 0, 62.5, 250, or 1000 mg/kg bw/day , 6 hours/day for 28 consecutive day s. Additional groups of 10 rats/sex were treated for the same time period at concentrations of 0 or 1000 mg/kg bw/day , followed b y a two - week recovery period. No dermal irritation was observed during treatment, and no treatment - related moribundity or mortality were found. No neurological effects were observed. There were no treatment - related effects on body weight, food consumption, ophthalmoscopic parameters, the estrus cycle, or effects on hematology , clinical chemistry , or urinaly sis parameters. There were no treatment relate d effects noted at necrops y on absolute or relative to body or brain organ weights, or microscopic treatment - related effects. **T he dermal toxicity NOAEL for male and female Wistar rats was 1000 mg/kg bw/day. A**  **LOAEL was not determined.**Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 32 of 52 This 28 - day

de rmal toxicity

study in the rat is **A cceptable** **/G uideline**  and satisfies the guideline requirement for a 28 - day

dermal toxicity

study (OPPTS 870.3200; OECD 410) in rats. **870.3465 90**  **- Day Inhalation**  **– Rat**  An inhalation study

is not required for an import tolerance. **A.4.2 Prenatal Developmental Toxicity**  **870.3700a Prenatal Developmental Toxicity Study -**  **Rat** In a developmental toxicity study (MRID 49040637 ), b enalax yl - M [ >99 % a.i.; batch # FCF/T/173 - 00 (ex 20493/57/B lotto II) ] was administered to 25 female Crl:CD (SD) BR rats/dose in 0.5% aqueous methylcellulose b y oral gavage at dose levels of 0, 10, 50, or 250 mg/kg bw/day

on gestation day s (GD) 6 through 15 (dose volume of 10 mL/kg bw/day). Dams were sacrificed and necropsi ed on GD 20. Pregnant uterus weight, numbers of corpora lutea, implantations, early

and late resorptions, and live and dead fetuses were recorded. Individual fetal and placental weights were recorded. All fetuses were sexed and subjected to gross extern al evaluations. Approximately half of the fetuses per litter underwent skeletal evaluations while the other half underwent visceral examinations. There was no evidence of maternal toxicity . No clinical signs, behavioral changes, body weight effects, or deaths were observed in dams during the study . An initial reduction in food consumption (GD 8 - 10) was observed in the dams at 250 mg/kg bw/day , but n o differences were noted for absolute body weight or corrected body weight gain . No gross pathological eff ects were noted at sacrifice, and no treatment - related effects on cesarean parameters were observed at any dose level. **The maternal lowest -**  **observed -**  **adverse -**  **effect level (LOAEL) for benalaxyl**  **- M**  **in rats was not determined. The maternal NOAEL was greate**  **r than or equal to 250 mg/kg bw/day.** There were no treatment - related effects on fetal sex ratio, fetal weight, post - implantation loss, resorptions per litter, or live litter size . Although the 10 and 250 mg/kg bw/day groups had increases in earl y resor

ptions, there was no dose response. Further, a slightly lower mean number of live fetuses per litter was noted in the 250 mg/kg bw/day group but was not considered treatment - related due to small magnitude (<1 fetus) , a corresponding lower number of mean i mplantations per dam, and the absence of a correlated increase in post - implantation loss. One to two malformed fetuses were present in all groups, including the control group. Therefore, no developmental effects were observed at doses up to 250 mg/kg bw/day . **The developmental LOAEL for benalaxyl**  **- M**  **in rats was not determined, and the developmental NOAEL was greater than or equal to 250 mg/kg bw/day.**  This developmental toxicity study in the rat is classified **Acceptable/Non -**  **Guideline ,**  but it does satisfy the guideline requirement for a developmental toxicity study (OSCPP 870.3700; OECDBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 33 of 52 414) in the rat . It is non - guideline because no significant developmental or maternal toxicity occurred at the highest dose level tested. However, a new study is not requi red at this time since a study at higher dose levels would not identify a more sensitive endpoint for risk assessment than the endpoint selected for risk assessment. **870.3700b Prenatal Developmental Toxicity Study -**  **Rabbit** In a developmental toxicity study (MRID 49040638 ), b enalax yl - M [ 97.14% a.i., batch # FCF/T/173 - 00 (ex 20493/57/B lotto II)] was administered to 24 mated female Himalayan rabbits/dose in 0.5% aqueous methylcellulose by oral gavage (dose volume of 4 mL/kg bw/day) at dose levels of 0, 50, 250, or 1000 mg/kg bw/day on gestation day s (GD) 6 through 27, inclusive. Dams were sacrificed on day 28 of gestation and underwent gross macroscopic examination. All fetuses were weighed, killed, sexed, examined for gross external abnormalities, and examined for both soft tissue and skeletal alterations. Heads from one - half of the fetuses were subjected to evaluation of the internal structures, including the eyes, brain, nasal passages, and tongue. Treatment with benalaxyl - M did not cause any maternal deaths, clinical signs, or effects on food consumption or body

weight/body

weight gain at doses up to and including 1000 mg/kg bw/day . **The maternal LOAEL for benalaxyl**  **- M**  **in rabbits was not determined. The maternal NOAEL was greater than or equal to 1000 mg/kg bw/day, the highest dose tested.**  No treatment - related fetal deaths or resorptions were noted, and fetal sex ratio was unaffected b y treatment. At 1000 mg/kg bw/day , there was a slight decrease (6%) in fetal body weight, correlated with a slight delay in the stage of development in this dose group as indicated by increased percentages of fetuses with incomplete chondrification of cartilaginous sternebra(e) 2, 3, and/or 4 compared to controls. However, these f indings were significant only when anal yz ed on an individual fetal basis, while the litter should be considered the statistical unit of measure. Skeletal abnormalities were limited to common sternebral abnormalities (including fused and abnormally

shaped sternebrae) and abnormalities of cartilaginous sternebrae and/or costal cartilages (including bipartite and/or abnormall y s haped cartilaginous sternebrae, fused or bifurcated costal cartilages). The combined incidence of these abnormalities was 2 (2), 3 ( 2), 3 (3), and 6 (6) fetuses (litters) in the 0, 50, 250, and 1000 mg/kg bw/day groups, respectivel y; the slight increase in the high dose group was not statistically significant or considered adverse. External and visceral abnormalities were incidental and not related to treatment and included one fetus in the 50 mg/kg bw/day group with an encephalocele and an open skull and two fetuses in the 1000 mg/kg bw/day

group: one with flexure of the right forelimb and one with minimal bilateral dilation of the lateral brain ventricles. **T he fetal LOAEL for**  **benalaxyl -**  **M in rabbits was not determined; and the fetal NOAEL**  **was greater than or equal to 1000 mg/kg bw/day.** The developmental toxicity study in the rabbit is classified **Acceptable/Guideline** and satisfies the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in rabbits .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 34 of 52 **A.4.3 Reproductive Toxicity**  **870.3800 Reproduction and Fertility Effects -**  **Rat** In a two - generation reproduction study (MRID 49040639), benalax yl - M (Benalax yl - M; 96.33% a.i.; Batch No. G 018/04) was administered to groups of 24 HanRcc:WI ST (SPF) rats/sex/dose in the diet at concentrations of 0, 120 [80], 360 [240], 1080 [725], or 3240 [2170] ppm, with lowered concentrations [those indicated in brackets] administered to lactating females and weaned pups to maintain a consistent intake of the test material in terms of mg/kg bw/day . Premating doses were respectivel y 0, 7.9, 23.5, 71.1, or 221.3 mg/kg bw/day for F 0 males, 0, 8.9, 26.7, 77.5, or 241.8 mg/kg bw/day for F 0 females, 0, 8.9, 27.1, 81.0, or 257.0 mg/kg bw/day for F 1 males, and 0, 9.9, 29.5, 85.8, or 280.2 mg/kg bw/day for F 1 females. Except for one high - dose F 0 “replacement” male, the F 0 and F 1 parental animals were administered the treated or control diets for at least 70 day s (for F 0 ) or 90 day s (for F 1 ) prior to mating, throughout mating, gestation, and lactation, and until necropsy after weaning of litters, and one litter was produced by each generation. The replacement animal was treated for onl y 55 days prior to mating. Additional groups of 24 F 1 pups/sex/dose were selected for post - weaning functional evaluations, including a functional observational battery /modified Irwin screen (FOB/modified Irwin) and locomotor activity

measurement. No significant treatment - related effects on mortality , clinical signs, body weight, or food consumption were noted in either sex or generation. Treatment - related increased incidences of hepatocellular h yp ertrophy (mainl y centrilobular) were observed in bo th sexes and generations at the high and high - mid doses and in low - mid - dose F 0 males, F 1 males, and F 1 females. Other findings included increased liver weights in high - dose males of both generations (absolute and relative - to - brain weights 20% and 16 - 17% greater than controls for F 0 and F 1 high - dose males, respectivel y; p<0.01), and increased incidence of gross enlargement of the liver in high - dose F 1 females (9/24 high - dose F 1 females vs. 0 controls; p<0.01). The liver is a target organ following benalax yl - M exposure, and these liver findings are consistent with other benalaxyl - M rat studies. Treatment - related decreased spleen weights were seen in high - dose females of both generations, with the absolute and relative (to brain) spleen weights 14% lower than controls for F 1 and 16% - 17% lower than controls for F 2 . Since hematology parameters and spleen histopathology were not examined as part of the current study , the toxicological significance of the decreased spleen weights is not known. A similar decrease w as not observed in the chronic rat study in either sex at comparable dose levels. No treatment - related effects were observed on fertility or reproductive performance in male or female rats in either generation. Estrous cycle length, sperm measures, repro ductive indices, precoital interval, gestation length, and postimplantation loss were not affected b y treatment. Significantly decreased numbers of primordial follicles and corpora lutea was observed in high - dose F 1 females (respectivel y - 26% and - 29%, re lative to controls); it is unknown whether this effect extended into the lower dose groups because the ovarian follicles were only counted in high - dose and control F 1 females. However, the magnitude of the deficit at the high dose suggests that any treatment - related effects at lower dose levels would not be toxicologically significant.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 35 of 52 **Under the conditions of this study, the parental systemic LOAEL for benalaxyl**  **- M**  **in female rats is 3240/2170 ppm (**  **241.8 mg/kg bw/day), based on decreased numbers of primordia**  **l follicles and corpora lutea in the F1 females, decreased uterus weights in F0 females, and a slight increase in the incidence of hepatocellular hypertrophy in both**  **generations. The parental systemic NOAEL in females is 1080/725 ppm (77.5 mg/kg bw/day).**  **The parental systemic LOAEL in males is 3240 ppm (221.3 mg/kg bw/day), based on increased liver weights and an increased incidence of hepatocellular hypertrophy in**  **both generation. The parental systemic NOAEL in males is 1080 ppm (71.1 mg/kg bw/day).** During postnatal day s (PNDs) 7 - 14 and 14 - 21, the body weight gain of the high - dose F 2 offspring of both sexes was significantly decreased ( - 11% to - 13%) with resultant significantl y decreased absolute body

weights on PND 14 (males: - 8%, p<0.05; females: - 1 0%, p<0.01) and PND 21 (males: - 10%; females: - 11%; p<0.01 for both). H igh - dose F 1 females had a treatment related 1.5 - day

delay in vaginal opening (p<0.05), with the group’s mean body weight at completion of vaginal opening comparable to that of the control group. Decreased absolute and relative (to brain) spleen weights were noted in high - dose males and females of the F 1 generation ( - 16% for both sexes and both parameters) and the F 2 generation (males: - 21% to - 22%; females:

- 17% to - 19%). Since hematol ogy parameters and spleen histopathology were not examined as part of the current study, the toxicological significance of the decreased spleen weights is not known. There were no effects on pup survival/mortality , litter size from birth to weaning, posti mplantation loss, viability indices, sex ratio, or male sexual maturation. There were no treatment - related clinical observations or gross findings in pups or weanlings. There was an increased incidence of hepatocellular hypertroph y in both sexes of F2 pup s at the high dose. Under the conditions of this study , postweaning functional investigations (FOB and locomotor testing) did not reveal any treatment - related effects. However, due to the absence of positive control data demonstrating competence of the te sting laboratory, the lack of provided information concerning the test methods and conditions, and the use of 15 - minute subsessions for the locomotor testing, the reviewer does not consider the apparent absence of neurobehavioral effects to be definitive. Since these parameters are not typically assessed in a reproduction study, and there are acute and subchronic neurotoxicity studies available to assess these parameters, these deficiencies do not interfere with the interpretation of this study. **The offsp ring LOAEL**  **for benalaxyl**  **- M**  **in rats is 221.3 mg/kg bw/day for males and 241.8**  **mg/kg bw/day for females, based on decreased pup body weight in F 2**  **pups of both sexes**  **and increased incidence of hepatocellular hypertrophy in F 2**  **pups of both sexes .**  **The offsprin**  **g NOAEL is 1080/725 ppm (71.1 mg/kg bw/day) for males and 77.5 mg/kg bw/day for females.**  Findings potentiall y indicative of reproductive toxicity included decreased ovarian follicle counts (primordial follicle s and corpora lutea) . The decrease in ovarian follicle counts in the high - dose F1 females is considered treatment - related and adverse. The results in the treated group were outside the historical control range, and the concurrent control values were generall y low compared to the historical control values. Although ovarian follicle counts were not assessed for the lower dose groups, it was concluded that the magnitude of the findings at the high dose are such that the results at lower doses would not be considered adverse.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 36 of 52 **T he reproductive toxicity L**  **OAEL for**  **benalaxyl -**  **M is**  **3240/2170 ppm ( 241.8 mg/kg**  **bw/day), based on decreased numbers of primordial follicles and corpora lutea in the F1 females. The reproductive toxicity NOAEL is 1080/725 ppm (77.5 mg/kg bw/day).**  This study

is **Acceptable/Guideline** and satisfies the guideline requirement for a two - generation reproductive study [OCSPP 870.3800; OECD 416] in the rat. Despite deficiencies in the conduct and reporting of this study and the occurrence of only minimal parental toxicity , offspring toxicity and potential reproductive toxicity were seen. **A.4.4 Chronic Toxicity**  **870.4100a (870.4300) Chronic Toxicity –**  **Rat** See under 870.4200a **870.4100b Chronic Toxicity -**  **Dog** In a 52 - week oral toxicity study (MRID 49040630 ) , four groups of beagle dogs (4/sex/group; 5.5 - 7.5 months of age) were administered via capsule 0, 10 (26 weeks)/300 (26 weeks), 30, or 100 mg/kg/day

(mg/kg) of benalax yl - M [96.33% (Batch No. G018/04] for 52 weeks . The 10 mg/kg/day

dose was increased to 300 mg/kg beginning week 27 to increase the chance of detecting toxic effects. The animals in each test group were subjected to clinical pathology , and gross and microscopic examinations. All animals survived to the end of the study. No clinical signs attributed to the test substance were observed at any dose. There were no treatment - related or toxicologically significant findings observed in the body weight data. No treatment - related effects were observed on hematology

values, clinical chemistry values, urinaly sis values, organ weight data, or during gross examination of tissues . Minimal or slight periportal fat vacuolation in the liver was observed in 3/4 males and 2/4 females from the 10/300 mg/kg/day dose group. All females from the 10/300 mg/kg/day

dose group had a minimal diffuse hy pertrophy of the thy

roid gland. **The lowest observed -**  **adverse effect level (LOAEL) for benalaxyl**  **- M**  **in beagle dogs was not**  **identified .**  This chronic toxicity

study in the dog is classified as **Acceptable/Guideline .**  **A.4.5 Carcinogenicity**  **870.4200a Carcinogenicity Study -**  **rat** In a combined chronic toxicity /carcinogenicity study

( MRID 49040634 ) , benalaxyl - M (96.33% a.i., L ot/Batch # G018/04) was administered in the diet to 20 male and 20 female Sprague Dawley CD Crl:CD(SD)BR rats at concentrations of 0, 60, 390, or 2535 ppm (0, 2.92, 19.0, and 126 mg/kg bw/day , respectively , for male rats and 0, 3.82, 24.6, and 167 mg/kg bw/day ,Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 37 of 52 respectivel y, for female rats) for 52 weeks (chronic study) and groups of 65 male and 65 female rats were administered the same dietary concentrations for 104 weeks (main carcinogenicity study). The equivalent doses for the main study were 0, 2.40, 15.7, and 104 mg/kg bw/day , respecti vel y, for male rats and 0, 3.13, 20.0, and 135 mg/kg bw/day , respectively , for female rats. No treatment - related, adverse effects were observed on mortality , clinical signs, detailed clinical observations, FOB parameters, body weight and body weight gain , hematology , nodules/masses, water consumption, food consumption, or eyes in either sex. Increased γ - glutamyl transferase (GGT) levels were observed in both sexes at 7000 ppm, with males displaying the greater effect. Both sexes showed slightly increased cholesterol values also. Absolute liver weight was not significantl y increased (11% - 12%) b y treatment with the test substance, but the liver/body weight ratio was significantly increased by

10% to 18% in high - dose male and female rats after 52 and 104 week s of treatment. The liver/brain weight ratio was not significantly affected by treatment with the test substance. Gross and microscopic examination showed treatment - related changes primarily in the liver and thy roid gland. Gross examination showed acc entuated lobulated pattern in the liver of 4/20 high dose male rats compared with none of the controls. In the chronic study (52 weeks), treatment related microscopic lesions in the liver included fatty change and hepatocellular hy pertrophy (primarily centrilobular) at the mid - and high - dose level in male rats, hepatocellular hypertroph y at the mid - and high - dose level in female rats, and fatty change and infiltrating inflammatory cells at the high - dose level in female rats. The incidences of bile duct hy perplasia and peribiliary fibrosis were decreased in high - dose male and female rats compared with that of controls. The incidence of th yr oid follicular cell hy pertrophy was significantly

increased in male rats in the mid - and high - dose groups and in femal es in the high - dose group compared with that of controls. In the main study

(104 weeks), the incidences of eosinophilic foci and spongiosis hepatis in the liver of male rats and biliary cy sts in females showed statistically significant positive dose relat ed trends but did not reach statistical significance at any dose; the incidence of eosinophilic foci was significantl y increased in females in the high - dose group compared with that of controls. The incidence of lesions in the thy roid gland was increased onl y in female rats in the high - dose group. These included follicular cell hypertrophy, follicular cell hy perplasia, and follicular ectasia. The incidence of ovarian stromal cell hy perplasia was increased in females at the high - dose level. **T he lowest**  **- obs**  **erved -**  **adverse -**  **effect level (LOAEL) for benalaxyl**  **- M**  **in rats is 2535 ppm**  **(males 104/females 135 mg/kg bw/day), based on an increase in GGT in males, increased triglycerides in males, increased cholesterol in both sexes, slight increases liver weight in**  **both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased**  **incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia**  **in females, and an increased incidence of ovarian stromal cell hyperplasia in females**  **. The no -**  **observed**   ** adverse** **- effect level (NOAEL) is**  **390 ppm (**  **males 15.7 mg/kg bw/day and females 20 mg/kg**  **bw/day )**  **.** At the doses tested, the incidences of thy roid follicular cell adenomas and follicular cell adenomas/carcinomas combined were marginall y increased in females at the high dose comparedBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 38 of 52 with that of controls. Additionally, there was an increased incidence of hepatocellular adenomas in both sexes at the high dose . This chronic/carcinogenicity study in the rat is **Acceptable/Guideline** and satisfies the guideline requirement for a chronic/carcinogenicity study ( O CS PP 870.4300 ; OECD 453) in rats. **870.4200b Carcinogenicity (feeding) -**  **Mouse** In a carcinogenicity study ( MRID 49040635), benalaxyl - M (96.33% a.i., Batch/L ot # G 018/04) w as administered in the diet to 50 male and 50 female CD - 1 (SPF) mice per dose at concentrations of 0, 200, 1200 or 7000 ppm for 78 weeks (equivalent to 0, 24.84, 156.05, and 962.23 mg/kg bw/day

for males and 0, 31.36, 196.72, and 1180.41 mg/kg bw/day for females). No treatment - relate d effects were observed on the following parameters: mortality , clinical signs, body weight, food consumption, and hematology parameters. The liver was identified as the target organ with treatment - related effects observed at 1200 ppm in male mice and at 7000 ppm in both sexes. At 7000 ppm, absolute liver weight, liver to brain weight ratio, and liver to body weight ratio were significant increased b y 52% - 63% in males and 52% - 61% in females compared with that of controls. Macroscopic examination showed a significant increase in liver nodules in males (18/50 vs 8/50 in controls, p<0.01) and in females 4/50 vs 1/50, N.S.) at 7000 ppm. At 1200 ppm, the only treatment - related finding in the liver was an increased incidence of diffuse hepatocellular hypertrop hy in male mice (21/50 vs 8/50, p<0.01). Treatment - related microscopic liver lesions were observed in male mice at 7000 ppm were diffuse hepatocellular hy pertrophy (49/50 vs 8/50 in controls, p<0.01), single cell necrosis (30/50 vs 10/50 in controls, p<0. 01), focal/multifocal necrosis (12/50 vs 5/50 in controls, p<0.05), and eosinophilic focus (6/50 vs 1/50 in controls, p<0.01). Treatment - related liver lesions were observed in female mice at 7000 ppm were diffuse hepatocellular hyp ertrophy (44/50 vs 0/50 in controls, p<0.01) and single cell necrosis (21/50 vs 5/50 in controls, p<001). In addition, the severity of the liver lesions also increased in both sexes at 7000 ppm, as evidenced b y the average severity grade and the incidence of mice with moderate a nd/or severe forms of the lesion . At 7000 ppm, one male and one female displayed thy roid follicular cell hyperplasia. **The lowest**  **- observed**  **- adverse**  **- effect level (LOAEL) for**  **benalaxyl -**  **M in male and female**  **mice is 7000 ppm (962.23 mg/kg bw/day for males and 1180 mg/kg bw/day for females) based on**  **increased liver weight in both sexes and an increased incidence of liver lesions**  **( diffuse hepatocellular hypertrophy in both sexes,**  **single cell necrosis in both sexes**  **, focal/multifocal necrosis in males**  **, and eosinophilic foci in males ). The NOAEL is 1200**  **ppm (156 mg/kg bw/day for males and 197 mg/kg/day for females).** At the doses tested, there was a treatment related increase in the incidence of hepatocellular adenoma in male mice, and an increase in the incidence of hepatocellular adenoma/carcinoma combined in male mice and female mice. The increased incidences exceeded those of historical controls for hepatocellular adenoma in male mice and hepatocellular adenoma/carcinoma combined in both sexes.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 39 of 52 This carcinogenicity

study in mice is **Acceptable/guideline** and satisfies the guideline requirement for a carcinogenicity study [OPPTS 870.4200; OECD 451] in mice . **A.4.6 Mutagenicity**  **Guideline No./ Study Type**  **MRID No. (year)/ Classification/Doses**  **Results Benalaxyl**  **- M**  870.5100 Bacterial reverse gene mutation (Ames Assay) 49040640 (2002 ) **Acceptable/guideline** 5 - 5000 μg/

plate +/

– S9 (plate incorporation) 5 - 15 0 0 μg/plate +/

– S9 (preincubation) **Negative** up to the limit dose; compound precipitation at ≥ 1500 μg/plate 870.5300 In vitro mammalian forward cell gene mutation (mouse ly mphoma cells) 4904064 2 (1999 ) **Acceptable/guideline** 5 - 7 5 μg/mL

– S9 1 5 - 100 μg/mL +S9 **Negative** up to cy totoxic concentration 870.53 75 In vitro mammalian chromosome aberration test [Chinese Hamster Ovary (CHO cells)] 4904064 1 (1999) **Acceptable/guideline Assay 1**  5 - 3000 μg/mL+/

– S9 ( 3

hrs.) **Assay 2** 5 - 150 μg/mL

– S9 ( 18

hrs.) 1 5 - 150 μg/mL + S9 ( 3 hrs.) **Negative** up to cy totoxic & insoluble concentration

870.5395 Mammalian ery

throcy te micronucleus assay

(mouse) 4904064 3 ( 2000 )

**Acceptable/guideline** 0, 100, 200, 400 mg/kg M 0, 75, 150, 300 mg/kg F Single i.p. injections Negative up to doses ( ≥ 300 mg/kg) causing overt toxicity

in preliminary

study (deaths and/or piloerection) **N -**  **malonyl -**  **N -**  **(2, 6 -**  **xylyl) -**  **D, L -**  **alanine** 870.5100 Bacterial reverse gene mutation (Ames Assay) 4904060 6 (2002) 33 - 5000 μg/plate +/

– S9 **Acceptable/guideline Negative**  up to the limit dose ; plate incorporation & preincubation 870.5300 In vitro mammalian cell gene mutation (mouse ly

mphoma cells) 490406 08 ( 2002 )

168.8

– 2700 μg/mL

– S9 (4 hrs.) 168.8

– 2 565 μg/mL+S9 (4 hrs.) 168.8

– 2700 μg/mL

– S9 ( 2 4 hrs.) **Acceptable/guideline Negative**  up to insoluble concentrations 870.5375 In vitro mammalian chromosome aberration test [Chinese Hamster Ovary (CHO cells)] 4904060 9 (2002)

**Assay 1** 60.3

– 1930 μg/mL - /+ S9

(4 hrs.) 60.3

– 1930 μg/mL - S9 ( 2 4 hrs.)

**Assay 2** Negative up to a cy totoxic concentration ( 1930 μg/mL - S9 , 24 &

46 hrs.)Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 40 of 52 **Guideline No./ Study Type**  **MRID No. (year)/ Classification/Doses**  **Results** 241.3

– 1930 μg/mL - S9 (4 6 hrs.) 241.3

– 1930 μg/mL+ S9 (4 hrs.) **Acceptable/guideline Methyl**  **- N**  **- malonyl**  **- N**  **- (2, 6**  **- xylyl)**  **- D, L**  **- alaninate**  870.5100 Bacterial reverse gene mutation (Ames Assay) 49040605 (2002) 33 - 5000 μg/plate +/

– S9 **Acceptable/guideline Negative**  up to the limit dose ; plate incorporation & preincubation 870.5300 In vitro mammalian cell gene mutation (mouse ly

mphoma cells) 49040607(2002)

156.3

– 2500 μg/mL+/

– S9 (4

hrs.) 156.3

– 2500 μg/mL+/

– S9 ( 2 4

hrs.) **Acceptable/guideline Negative**  up to insoluble concentrations

870.5375 In vitro mammalian chromosome aberration test [Chinese Hamster Ovary (CHO cells)] 490406 10 (2002)

**Assay 1** 19.5

– 2500 μg/mL - /+S9

(4 hrs.) **Assay 2** 6 25

– 1 875 μg/mL - S9 (24

& 46 hrs.) 625

– 2500 μg/mL + S9 (4 hrs.) **Assay 3** 1000 - 1500 μg/mL - S9 (24 & 46 hrs.) **Acceptable/guideline Confirmed positive:**  **Assay 2** 937.5, 1250 & 1875 μg/mL - S9 (24 hrs.) & 937.5 μg/mL - S9 ( 46 hrs.) . **Assay 3** 1500 μg/mL - S9 (24 hrs.) 870.5375 In vitro mammalian chromosome aberration test (Human l ymphocy

tes) 4904061 1 (2002)

**Assay 1** 0.078

– 10mM - /+S9 ( 3

hrs.) **Assay 2** 0.313

– 10 mM - S9 (2 0

hrs.) 0.313

– 10 mM +S9 ( 3 hrs.) **Acceptable/guideline P**  **ositive:** at 10 mM - S9 (20 hrs.) Limit dose

870.5395 Mammalian ery

throcy te micronucleus assay

(mo use) 49040612 (2002)

0, 500, 1000, 2000 mg/kg M & F Single i.p. injections **Acceptable/guideline** Negative up to an overtly toxic dose ( 2000 mg/kg)Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 41 of 52 **A.4.7 Neurotoxicity**  **870.6100 Delayed Neurotoxicity Study -**  **Hen** A delayed neurotoxicity

is not required and is not available. **870.6200 Acute Neurotoxicity Screening Battery** In an acute neurotoxicity

study (MRID 49040626) , groups of unfasted, 6 - to 9 - week - old CRL : Wistar rats (12/sex/dose) were given a single oral (gavage) dose of b enalaxyl - M ( 96.53% w/w; Batch/Lot #P/10/106) in 0.5% aqueous carbox ym ethylcellulose (CMC) solution (dose volume of 10 mL/kg) at doses of 0, 500, 1000, or 2000 mg/kg bw and observed over a 14 - day postdose period. In addition to obser vations of all animals for clinical signs of overt toxicity conducted daily from the day

of dosing to study

termination on Day

15, weekly

body weight measurements and neurobehavioral assessments (functional observational battery , landing foot splay testing , grip strength measurements, and motor activity testing) were conducted on all animals on pre dose Day - 7, dose Day

0 (starting at approximately 30 minutes after dosing), and post - dose Day s 7 and 14. At study

termination on Day

15, at least 6 animals/sex/group were perfusion - fixed. Other surviving animals were euthanized under pentobarbital anesthesia by exsanguination. Gross necrops y was performed on each animal. Brain weights of non - perfused animals were obtained on Day

15; brains of perfused animals were weighed on Day 17, after a 48 hour period of post - perfusion whole body fixation. C entral nervous sy stem and peripheral nervous s ys tem tissues from the control (0 mg/kg) and high dose (2000 mg/kg) groups of perfused animals (6 animals/sex/group) were proc essed into slides (embedded in paraffin or plastic Epoxy resin and sectioned) for histopathological evaluation. There were no treatment related effects on mortality , clinical signs, body weight, brain weight or gross and histologic pathology or neuropath ology . FOB, landing foot splay, fore - and hindlimb grip strength, and motor activity testing revealed no treatment related effects. **Based on the absence of treatment related effects in this study, a LOAEL was not identified.**  **The NOAEL for benalaxyl**  **- M**  **te chnical product was 2000 mg/kg bw**  **administered orally in adult male and female Wistar rats.** This neurotoxicity

study is classified as **Acceptable/Guideline** and satisfies the guideline requirement for an acute neurotoxicity study in rats (OCSPP 870.6200a; OECD 424). **870.6200 Subchronic Neurotoxicity Screening Battery** In a subchronic neurotoxicity study (MRID 49143501 ), benalax yl - M technical product (Benalax yl - M; 96.53% a.i.; Batch/Lot #P/10/106) was administered in the diet to 12 CRL :Wistar rats/sex/group at dietary

levels of 0, 1000, 3000, or 10,000 ppm (resulting in calculated daily dose levels of 0, 67, 206 and 784 mg/kg bw/day for males and 0, 78, 325, and 1139 mg/kg bw/day for females, respec tivel y) for 90 days. Treatment was initiated on Day 0, and the neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) was performed in all animals on Days - 7, 14, 42, and 77 (Weeks - 1, 3, 7 and 12). At studyBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 42 of 52 termin ation (Day

90), at least 6 animals/sex/group were euthanized via in situ perfusion fixation , and the central and peripheral nervous sy stem tissues from 6 male and 6 female perfused control and high - dose animals were subjected to histopathological evaluatio n. Additional measures included ophthalmoscopic examination (all animals on Day - 6 and control and high - dose at term), serum GGT activity (in all animals at term), gross pathology (of all surviving animals), and brain and liver weights (all surviving anim als). There were no treatment - related effects on mortality , general or detailed clinical signs, brain weight, or macroscopic and neurohistologic evaluations. Neurobehavioral test measures including FOB, landing foot splay , grip strength and motor activity testing also revealed no treatment - related effects for either males or females at any dietary level. Absolute body

weights and food consumption were not affected b y treatment in either sex. Liver we ights (absolute and relative) were significantly increased in non - perfused males at the low - , mid - , and high - dose treatment levels (absolute weights: +29%. +42%, and +51%, respectively ; p<0.05 or p<0.01) and in non - perfused mid - and high - dose females (abso lute weights: +20% and +45%; p<0.01 for high - dose, onl y) . In perfused animals, liver weights were increased only at the high - dose treatment level (both sexes), but it is possible that liver weights of these animals were influenced by the perfusion/fixatio n process. Increased serum GGT activity was seen in high - dose animals of both sexes (males: 14.0 U/L ; females: 16.1 U/L; both vs. 5.0 U/Lfor controls and p<0.01), which correlated with the increased liver weights in the high - dose animals of both sexes. **Based on increased serum GGT activity in both sexes and increased absolute and relative liver weights, the LOAEL for**  **benalaxyl -**  **M in rats is 10000 ppm (equivalent to 784 mg/kg**  **bw/day for males and 1139 mg/kg bw/day for females). The NOAEL is 3000 ppm (equi** **valent to 206 mg/kg bw/day for males and 325 mg/kg bw/day for females).** The study is classified as **Acceptable/Guideline** and satisfies the guideline requirement for a subchronic neurotoxicity

study in rats [OCSPP 870.6200b; OECD 424]. **870.6300 Develop mental Neurotoxicity Study**  A developmental neurotoxicity study is not required and is not available.Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 43 of 52 **A.4.8 Metabolism**  **870.7485 Metabolism**  **- Rat**  In four metabolism studies (MRIDs 49040644, 49040645 and 49040646, 49040647), [ 14 C - U aniline ring] - benal ax yl - M (>99% a.i., Batch/lot No. - MRID 49040644: FCF/T/155 - 98, MRID 49040645: 20376/76, MRIDs 49040646 and 49040647: B01/L - 4 ) was administered to groups of 5 male Sprague Dawley

rats at doses of 0 or 10 mg/kg bw or to groups of 2

– 4 male and female Sprague Dawley

rats at doses of 10 or 100 mg/kg bw by oral gavage. (MRIDs 49040644 and 49060445). Following a single 10 mg of 14 C [ U - aniline ring ]

– benalax yl - M/kg bw oral dose to male rats, the test material was readil y absorbed and excreted mainly through the fecal route (~85%) and to a smaller extent via the urine (~8%) within 48 hours. Biliary

excretion, as demonstrated in the subsequent studies, explains the high elimination of absorbed radioactivity via the fecal route. The highest tissue concentrations, as percent of administered dose 0.5 hours after treatment, were found in the intestinal wall (8.0%), liver (8.3%), and stomach wall (6.4%). Total radiolabel recovered in the tissues 0.5 hours after treatment was 23%. By

72 hours after treatment, all tissue concentrations were <1% of the administered dose. The pharmacokinetic profile was characterized by a peak level between 10 minutes (fir st sampling time) and 1 hour after admin istration. After peaking , a gradual decline of radioactivity

was observed leading to an elimination half - life (t 1⁄2 ) of ~ 18 hours. C max was 452 ± 158 ng eq./m L while AUC 0 - ∞ was 6593 ± 470 h our • ng eq./mL . The average MRT (mean residence time extrapolated to infinity) was 25 ± 4 hours. Excretion in the expired air was negligible ( <0.1%). No residual radioactivity was found in the carcasses 168 hours after treatment. The total excretion accounted for 93.4% of the administered dose. Benalax yl - M was extensively

metabo liz ed , principally

by oxidation of the methyl group on the aniline ring to the hydrox ym ethyl and finally to the carboxylic acid. Minor metabolic pathway s were the hydrox yl ation of the phenyl ring and the h ydrol ys is of the carbox ym eth ylgroup. (MRIDs 49040646 and 49040647). The pharmacokinetics of total radioactivity in blood, plasma, tissue, urine, feces, bile and expired air following single oral dose of 10 mg/kg of 14 C - [ U - aniline ring] - benalaxyl - M was investigated in male and female Sprague Dawley rats. Elimination of absorbed radioactivity was mostly

through the feces. Biliary excretion was substantial for both sexes (80.5% of dose male; 62% of dose females within 72 hours), indicating most of the radiolabeled was absorbed and then excreted via the bile into the feces. Female rats excreted approximately

double the amount of radiolabel into the urine (7.61% of dose males; 17.23% of dose females). The highest tissue concentrations were f ound in the liver and GI tract of both male and female rats. The amount of radiolabel recovered in the tissues was approximately

double in males (17.29% of dose) relative to females (7.96% of dose) 1 hour after treatment. Thereafter, tissue concentratio ns were <1% for both male and female rats at 72 and 168 hours. Total recovered radioactivity was high, 99% in the male and 97% in the female, being most of the radioactivity was eliminated within 48 hours post dosing. Negligible levels of radioactivity

( <0.03%) were recovered in the expired air. A fter oral administration of 10 mg/kg bw 14 C - benalax yl - M to male rats, the blood C m ax was 1.22 ± 0.45 μg eq./g at tmax 1.8 hours post dos e. The radioactivity

was detectable up t o 120 - 144 hours, resulting in a t 1⁄2 ,z (apparent terminal half - life of total radioactivity) of 62.4 hours. The AUC 0 - ∞ (area under the plasma concentrationBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 44 of 52 versus time curve up to infinite time) to the total radioactivity was 17.8 ± 1.9 μg eq. ∙hour/g . After oral admin istration of 10 mg/kg bw 14 C - benalaxyl - M to female rats, C max was 1.93 ± 0.49 μg eq./g at 1.8 hours post - dose . T he levels of radioactivity declined with a t 1⁄2,z of 80 hours . The AUC 0 - ∞ was 34.3 ± 5 . 8 μg eq. ∙hour/g . After oral administration of 14 C - benalax yl - M at a dose of 100 mg/kg bw to male rats, C max was 5.44 ± 2 . 25 μg eq./g at 3.3 hours post - dos e. Detectable levels of radioactivity were measured until 96 - 120 hours post - dos e , with an average t 1⁄2 ,z of 102 hours. The AUC 0 - ∞ was 155 . 9 ±26 μg eq. ∙hour/g . After oral administration of 100 mg/kg bw 14 C - benalax yl - M to female rats, total radioactivity

in blood reached a C max of 8.5 ± 3.21 μg eq./g at 6 hours post - dos e . The levels of radioactivity

declined with a t 1⁄2 ,z of 59 h ours . The AUC 0 - ∞was 212.3 ± 71.2 μg eq. ∙hour/g . The data suggest that absorption and elimination were not saturated as there was not a 10 - fold increase in C max or tmax . Parent 14 C - benalax yl - M was excreted exclusively in the feces as a minor component and accounted for 1.49% in female s and 2.94% in males . No parent compound was found in the urine. 14 C - benalax yl - M was extensively metabolized by

oxidation of the methyl group on the aniline ring to h ydrox ym ethyl and finally to carboxylic acid. Other metabolic pathway s were hydrox yl ation and di - hydroxylation (followed by methylation) of the phenyl ring. Conjugation of hydrox ylgroup with glucuronic acid was also found. Total identified radioactivity accounted for 82.11% and 87 . 69% of the administered dose in the excreta of male and femal e rats respectivel y. The above studies are well conducted and complement each other. Together, these metabolism studies are classified **Acceptable / Guideline** and satisfy the guideline requirement for a metabolism study [ OCSPP 870.7485, OECD 417] in the rat. **870.7600 Dermal Absorption**  **- Rat**  A dermal absorption study is not required and is not available. **A.4.9 Immunotoxicity**  **870.7800 Immunotoxicity**  In an immunotoxicity

study (MRID 49040648), benalaxyl - M technical product (purity 96.53%, Lot No. P/10/106), was administered in the diet for 28 consecutive day s at concentrations of 0, 1000, 3000, or 10,0 00 ppm (equivalent to 0.0, 80.5, 250.4, and 924.6 mg/kg bw/ day , respectivel y) to groups of 12 male Crl: WIrats/dose. A concurrent cyclophosphamide (CPS) positive control group of 12 male Wistar rats received 25 mg CPS in saline/kg bw/day by IP injection from day s 22

– 27. The rats were inspected for signs of morbidity and mortality twice daily . General c linical observations were made at least once a day while d etailed clinical obser vations were made weekly . Body weight and food consumption were determined twice weekly while water consumption was measured dail y. All rats received a single IV injection i nto the tail vein of sheep red blood cells (SRBCs, 2×10 8 cells/rat) on Day 22. The rats were sacrificed following an overnight fast and blood collected for an enzy me - linked immunosorbent assay forBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 45 of 52 anti - SRBC IgM antibody and gamma glutam yltransferase (GGT ) activity . All rats were necropsied and observed macroscopically . Histopathology was not done. No treatment - related effects were found on body weight, food consumption, food efficiency , or water consumption. No clinical signs of toxicity were observ ed, and all rats survived until scheduled sacrifice. There was a slight increase in GGT activity (6.5±1.7 U/Lvs 5.0±0.0) in rats receiving 10,000 ppm, however, the significance of this result is obscured by the low sensitivity of the assay

at the low end of the linear range. No treatment - related effects were noted at necrops y. The absolute liver weights of mid - and high - dose rats were statistically increased 17% and 39%, respectivel y, while the liver weight relative to body weight of low - , mid - , and high dose rats were statistically increased 10%, 23%, and 48%, respectively . In addition, the relative to body kidney weight of high - dose rats was statistically increased 8%. While the increased absolute and relative to body liver weights were observed, no histopathology was done. The liver effect was a common observation of this chemical and was considered treatment - related. **The systemic LOAEL**  **for male Wistar rats following dietary treat**  **men t for**  **28 days with**  **benalaxyl -**  **M was 3000 ppm (250.4**  **mg/kg bw/day ) based on the increases in absolute and**  **relative to body liver weights. The NOAEL was 1000 ppm (80.5**  **mg/kg bw/day).** Dietary treatment of male rats with 1000, 3000, or 10,000 ppm benalax yl - M technical product for 28 continuous day s did not affect spleen, thy mus, or mesenteric l ymph node weights or decrease the anti - SRBC IgM immune response. T here were no statisticall y significant dif ferences in anti - SRBC IgM levels in treated groups when compare to the vehicle control group. High inter - individual varia bility was noted in all the treatment groups as well as in the control group. Evaluation of the individual animal data of this study did not show any

trend or distribution that would demonstrate significant suppression of anti - SRBC antibody response. Posi tive control group had statistically significant d ecrease in the anti SRBC IgM levels. This confirmed the ability

of the test sy stem to detect immuno - suppressive effects and confirmed the validity of the study design. The Natural Killer (NK) cells activity was not evaluated in this study . The toxicology database for b enalaxyl - M does not reveal any evidence of immunotoxicity . The overall weight of evidence suggests that the chemical does not directly target the immune s ys tem. Under HED guidance a NK cell activity assay is not required at this time. **T he immunotoxicity NOAEL for male Wistar rats treated for**  **28 consecutive days wi**  **th benalaxyl**  **- M**  **was 10,000 ppm (924.6 mg/kg bw/day). A LOAEL was not**  **established .**  This immunotoxicity

study is classified **Acceptable / Guideline** and satisfies the guideline requirement for an immunotoxicity study ( OCSPP 870.7800) in the rat. **A.4.10 Other**  Non - guideline Mechanistic In a 14 - day mechanistic study ( MRID 49040636), benalax yl - M (96.53% a.i.; L ot/Batch # P/10/106) was administered in feed to groups of 18 male Crl: CD - 1(Icr) Br mice at dietaryBenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 46 of 52 concentrations of 0, 200, 1200, or 7000 ppm (0, 30.9, 184.4, and 1094.1 mg/kg bw/day, respectivel y). Another group of 18 male mice were administered phenobarbitone (PB) in the diet at 850 ppm (139.6 mg/kg bw/day). Six mice/groups were sacrificed for evaluation of cell proliferation after treatment for 3 and 14 day s (Allocation A and B animals, respectivel y). Allocation A and B animals were implanted subcutaneously with osmotic mini - pumps containing bromodeox yuridine (BrdU) on days 6 and 8 of acclimatization, respectivel y. Six mice/group were sacrificed after treatment for 14 day s for evaluation of liver microsomal enzy me activity , mRNA ge ne expression, and oxidative stress (Allocation C animals). No effects were observed on mortality , clinical signs, body weight, or body weight gain, in mice administered benalaxyl - M or PB. Absolute liver weight, liver/body weight ratio, and liver/brain weight ratio were increased 16% to 17% and 15% to 20% in mice treated with benalax yl - M for 3 day s or 14 day s, respectively , and absolute and relative liver weights in mice treated with PB were increased b y 32% to 36% and 33% to 41% after 3 and 14 day s, re spectively . No benalaxyl M - or PB - related macroscopic lesions were observed in this study . Centrilobular to diffuse hepatocellular h yp ertrophy were observed in 0/6, 1/6, 2/6, and 4/6 mice treated with 0, 200, 1200, or 7000 ppm of the test substance, resp ectively , for 3 day s and 0/6, 2/6, 4/6, and 3/6 mice, respectivel y, after 14 days. All six mice treated with PB for 3 or 14 day s had centrilobular to diffuse h ypertroph y. There was a dose - related increased severity of the lesions in mice treated with ben alax yl - M , and the lesions in PB - treated mice was more severe than in those treated with IR6141. Analy sis of the number of BrdU - positive nuclei in hepatocy tes as an indicator of cell proliferation showed that cell proliferation was increased at all doses of IR6141 after 14 day s; the data was equivocal after treatment for 3 day s. PB treatment resulted in an increase in cell proliferation after 3 and 14 day s. The effect of benalaxyl - M and PB treatment was anal yz ed on following microsomal liver enzy mes: 7 - ethox yr esorufin O - dealkylase (EROD, Cy p1a1 isoenzy me), 7 - methoxy

resorufin O - dealkyla se (MROD, Cy p1a2 isoenz ym e), 7 - pentox yresorufin O - dealkyl a se (PROD, Cy p2b1 ), testosterone 6β - hydrox yl ase ( 6β - TOH, Cy p3a), lauric acid 12 - hydrox yl ase (LA12OH, Cy p4a1 ), and lauric acid 11 - hydrox yl ase (LA11OH, Cy p2e1 ). EROD, MROD, and LA12OH activities were slightly increased at 1200 and 7000 ppm; PROD, 6β - OH, and LA11OH activities were moderatel y increased at 7000 ppm; and PROD activity was moderatel y increased at 120 0 ppm and 6β - OH and LA11OH activities were slightly increased at 1200 ppm. Treatment with PB for 14 day s caused moderate increases in EROD, MROD, 6 - β TOH, and LA11OH activities, a pronounced increased in PROD activity and a slight increase in LA12OH activ ity . The expression of the Cyp1A - 1 , Cyp1A - 2 , Cyp2B10 , and Cyp3A11 genes were increased at 7000 ppm and the expression of Cyp1A - 1 , CypB10 , and Cyp3A11 were increased at 1200 ppm. The expression of Cyp2B10 was increased 45.8 - and 195.7 - fold at 1200 and 700 0 ppm, respectively , and the expression of Cyp3A11 was increased 15.58 - fold . Treatment with PB for 14 day s caused increased expression of all the genes tested. Examination of the parameters of oxidative stress, reduced glutathione (GSH) and oxidized glut athione (GSSH) in liver, and thiobarbituric acid reactive substance (TBARS) in liver and serum, showed no effect of treatment with benalax yl - M or PB for 14 day s. This 14 - day mechanistic study in the mouse is **Acceptable/Non -**  **guideline.** The study provides an assessment of possible liver toxicity for use in the evaluation of the mode of action of hepatocellular tumor induction in mice by benalaxyl - M .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 47 of 52 **Appendix B. M**  **etabolism B.1**  **Metabolism Summary Table Table**  **B. 1. Tabular Summary of Metabolites and Degradates**  Chemical Name (other names in parenthesis) and Structure Matrix Percent TRR (PPM) Matrices - Major Residue (>10%TRR) Matrices -

Minor Residue (<10%TRR) Benalax yl - M Grape - Fruit 26.4 % (0.023) NA Tomato - Fruit 43.5 % (0.025) NA Lettuce 41.5 % (0.84) NA Rat NA 3% feces ♂ 2% feces ♀ R5 N - (h ydrox yphenyl acetyl) -N - (2 - hydrox ym ethyl - 6 - methylphenyl) - alanine Grapes - Fruit NA NA Tomato - Fruit NA NA Lettuce NA NA Rat 22% feces ♀ 3% feces ♂

R6 methyl -N - (phenyl acetyl ) -N - (2 - carbox y6 - hydroxy

methylphenyl) - alaninate Grapes - Fruit NA NA Tomato - Fruit NA NA Lettuce NA NA Rat 11% feces ♂ 4% feces ♀Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 48 of 52 **Table B.**  **1. Tabular Summary of Metabolites and Degradates** Chemical Name (other names in parenthesis) and Structure Matrix Percent TRR (PPM) Matrices - Major Residue (>10%TRR) Matrices -

Minor Residue (<10%TRR)

R7 (methyl - N - (h ydrox yphenylacetyl ) -N - (2 - carbox y- 6 - methylphenyl) - alaninate Grapes - Fruit NA NA Tomato - Fruit NA NA Lettuce NA NA Rat 24% feces ♀

8% urine ♀ 38% feces ♂ 1% urine ♂ G10, T4, L4 Grapes - Fruit 11% (0.0093) NA Tomato - Fruit NA 3.2% (0.0018) Lettuce 11% (0.23) NA Rat NA NABenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 4 9 of 52 **Table B.**  **1. Tabular Summary of Metabolites and Degradates** Chemical Name (other names in parenthesis) and Structure Matrix Percent TRR (PPM) Matrices - Major Residue (>10%TRR) Matrices -

Minor Residue (<10%TRR) G13, [T9, T11], [L 3, L5 - L9, L11] Grapes - Fruit 14% (0.012) NA Tomato - Fruit NA 5.3% (0.0030) Lettuce 10% (0.21) NA Rat NA NA

G15, T10, L 10 Grapes - Fruit 11% (0.0097) NA Tomato - Fruit 32% (0.018) NA Lettuce 30% (0.61) NA Rat NA NABenalax yl - M Human Health Risk Assessment DP No. D 416044

Page 50 of 52 **B.2 Metabolic Pathway s** 

**Proposed Metabolic Pathway of Benalaxyl**  **- M**  **for Grape**  **s** The figure below was copied without alteration from MRI D 49040680 .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 51 of 52 **Proposed Metabolic Pathway for the Rat.** The figure below was copied without alteration from MRI D 49040647 .Benalax yl - M Human Health Risk Assessment DP No. D 416044

Page 52 of 52 **Appendix C. Physical/Chemical Properties**  **TABLE C**  **. 1.**  **Physicochemical Properties of the Technical Grade Test Compound: Benalaxyl**  **- M**  **.**

Parameter Value Reference Melting point/range 74.86 °C

D421271, S. Mathur, 22 - JUL - 2014 Boiling point 330 °C pH 7.1 Density 1.19 ± 0.022 g/ml (19.5 °C) Water solubility (at 25 °C) 37 .0 mg/L Solvent solubility (g/L) n - heptane - 17074 mg/L xylene - >39 (%w/w) acetone - >49 (%w/w) ethyl acetate - >49 (%w/w) 1,2 - dichloroethane - >50 (%w/w) methanol - >50 (%w/w) Vapor pressure at 25 °C 6.7 x 10 - 4 Pa Dissociation constant (pKa) Not applicable Octanol/water partition coefficient Log(KOW) 3.54 at 20 °C UV/visible absorption spectrum 202 nm - 3.4 x 10 4 ξ (dm3 /(mol x cm)) 254 nm - 4.6 x 10 2 ” 259 nm - 4.6 x 10 2 ” 265 nm - 6.1 x 10 2 ” 275 nm - 4.4 x 10 2 ” **Appendix D. Review of Human Research** This risk assessment does not rely on data from studies in which human subjects were intentionally

exposed to a pesticide or other chemicals .