### Halauxifen-methyl l l uman H ealth Risk Asse ss ment DP No. D4 0 6145



### UNITED ST ATES ENVIRON MENT A L PROTECTlON AGEN CY WASHIN GTON, D

. C

. 20460
OFFICE OF CHE MICAL SAFETY AND POLLUTION PREVENTION
MEMORANDUM
Date: 

### March 21, 20 16
SUBJEC T: 

### Hal a u xifen-met h y l

- New Act i ve In g r e

di en t Hum a n H ea l th Ri sk As sess ment for P roposed Uses on Ce r eal Grain s ( B a rl ey, Wheat, and Tr itical e).

FROM: PC Code: I 1750 I Decision No.: 470 1 69
P

etition No.: 2£ 8086 Ri sk Assess m ent Type: Si n g l e Chemical Aggregate TXR No.: NA M RI DNo.: A DP Barcode: D40 6 I 45 Re g i s tration No .: 627 19-A AE, AAG, AAN , AAR , AAU Regulatory Actio n : Section 3 Case No.: Non e CAS No.: 94383 1- 98 -9 40 Cf' R: New Active In g r edie nt


### E li zabe th Holman , DrPH , Ph ys i ca l Scientis t

### ~**~** Linn ea Han se n , Ph.D. , Bio lo g i st

### 0~').**~** 

## frv D ouglas D otso n. Ph.D ., Se nior C hemi s t 

### IJ..**t&"'~** 

# \;

A 

### Za id a F i g u eroa, D rPH , Indu s tri al H yg i e ni s t

### p~r-J??~/:_ Ri s k Assess ment Branch II (RA B II ) 

# "r Health Effects Di v i s ion ( HED ; 7509P)
THROUGH: 

### C hri sti n a Swa rt z, B r anch Chief RAB II

# c~ H ED (7509 P )


### Charles W. 

### S mith fll , 

### Branch C hief. RAB I 

# Q

.

# ~ El i zabet h Me nd ez, Ph.D ., Senio r Toxicolo g i st

# ~ Ri sk Assessment R ev i ew Comm itt ee HED (7509 P )
TO: 

### Dominic Sch ul e r , Risk Mana ge r R ev i e\.v er Reub e n Bari s, Ri s k Man age r ! Produ ct Manager 25 

### Dan Kenny , Bran c h Chief H

erb i c id e B ran c h R

egistration Division (7505 P )



### Pa ge 1of1 26Halauxifen-methyl H uman Hea l th Risk Assessment D P No. 0406145

1

. 0 EXECUTIV E SUMMARY

. .. ......... ... .......... .... ........... ... .......... .. ............ .. .. . ...... . . .. . .. . . . . .. . . . . .. . . . . .......... 4 2.0 H E D RE COMME DATIO 

### s
......................................................................................................... 7 2.1 Data Deficiencies / Conditions of Registratio n .
......... .... .......................... .. ........... . . . ............ 7 2.2 To l erance Considerations

............................. ... ............ . ........... . ............... . .. . .... . . .. . . . . . . . . . . .. . . .. 7 2.2.1 Enforcement Analytical Method

....... . ........................................ . ............ . ............... 7 2

. 2.2 International Ha1moni za tion

................................................................................... 8 2.2.3 R ecommended To l erances
............. ... .............................................. . ....... . . . . . ........ . . . 8 2.2.4 Revisi o n s to Petitioned- For Tolerances

.................................................................. 9 2.3 Label Recommendations

.................... . ........ .. .... . ............................................................... 10 3

. 0 I NTRODUCTION

. .. . ... . .. .. ....................... . ............................... .. ... . ....... .. .. . ..... .. . . .. . .... . . .... . . ..... . . . ... 10 3.1 Chemical Identity

. .. ................................................................................ . . . ....... . ................ 10 3.2 Physical / Chemical Characteri s tic s
.. .. .... .. ... . ............................................. . ......... . . . ............ 11 3.3 Pesticide Use Pattern

....... .. ...... ....... . ............... . ........... . ... . ........ .. .. . ........ . .. . . . .. . .... . . .. ....... . ... . 11 3.4 An t icipated Exposure Pathways

............................................... . ....................................... 12 3.5 Co n sidera ti on of Environmental Justic e
.... .. .... . ...... .. ... . .................................................... 13 4.0 H AZARD CHARACTERIZATION A D DOSE-R ES PONSE ASSESSMENT

.............................. . .......... 13 4.1 Toxicology Studies Available fo r Analysis

............................... . ............ . ......... . . .. ........... . 14 4.2 Absorption, Dist r i b ution , Metabolism, & El i m i nation (ADME)

........ ..... . . .... . . .. . . . . ........ . . . 15 4

. 2.1 Derma l Absorption

.. ..................................................... . ........................... . .......... . . 1 6 4.3 Toxicological E ffects

........ . ........................................ .. ........................... . ............ .. ........... 17 4.3.2 Mode of Action for Liver Toxicity of H alauxifen-Methyl

........ . .. . .... . . . .. . . . . . . . ...... . 20 4.4 Safety factor for Infants and Chi l dren (f.'QPA Safety Factor)

. .. . .. ........ .... .... . .... . . . . . ........ . . 23 4.4.1 Completeness of the Toxicolog y Database

.... .... .......... .. ............. .. ........................ 23 4.4.2 Evidence of Neurotoxicity

.. . . . ........... .... .... . .... .. ... .. ...... .. .. . ........ . .. . .. . .......... . ........... 23 4.4.3 Evidence of Sensitivity / Susceptibi l ity i n the D eve l oping or You n g A ni ma l

....... 23 4.4.4 Residual U ncertaint y in the Exposure Database

................................................... 24 4.5 Toxicity Endpoin t and P oint of Departure Se l ections

. .. ... . ....... . ........................... . ...... . .... 24 4.5. 1 Dose-Response Assessment

.. ........... .... ......... ... ........... . ............... . ......................... 24 4.5. 2 Recommendation for Combi nin g Routes of Exposures for Risk Assessment..

. . . . 26 4.5.3 Cancer Classification and R i sk Assessment R ecommendation

......... . .... . . . ... . .. . . . . . 27 4.5.4 Summary of Points of Departure and T ox i c i ty Endpoints Used in H u man R isk Assessn1ent.

................................................................................................ . . . . . .......... . . 27 5.0 D I ETARY EXPOSURE A D R I SK ASSESSMENT

.. ..... ......... .. .. .... ...... .. . ... . .. ........ . .. . ............. . .......... 30 5.1 Metabolite/Degradate Residue Profile

.......... .. .......... .. ............ . .. .. ........... . ....................... . . 30 5

. 1.1 Summary of Plant and A nimal Metabolism Studies

................... . ......................... 30 5

. 1

. 2 S u mma r y of Environmental Degradation

.. .. ... .. .. ....... .. .. . ................................. . ... . 31 5

. 1.3 Comparison of Metabolic Pathways

......................... .. .. . ......... . .. . ............ . . . . . ......... 32 5.1.4 Residues of Concern Summary and Rationale

. . ................................. . . . . .. . . ......... .. 33 5.2 Food Residue P rofi l e
... ... .. .. ............. ... ...... ... ... ... ..... .. .. ... ... . ....... . ........................................ 34 5.3 Water Residue Profi l e
................................................ . ........... .. ............ . . .. ........... . . . ........... 35 5.4 Dietar y Risk Assessment

............... . .............. . ................................................... . . . . . .......... . 35 5.4.1 Descript i on of Residue Data Used in Dietary Assessment..

... .. . .. . . .. . ........ . ..... . ..... 36 5.4.2 Dietary Exposure Risk Es timat es
... ... .......... ... .. .. ....................... . ........ . . . . .. . . .......... . 36 5.4.3 Dietar y Risk Assessment Summary Table

................................. . .......................... 36 6.0 R ES IDE NT IAL (NON-0CCUPATl0 1 AL) EXPOSURE / RI SK CllARACTERIZJ\TION

............... . ......... . 37 6.1 Residential Bystander Post-Application I nha l at i o n Expo s ure
.. . . ................... . ... . .. . ......... . . 37

Page 2of 1 26Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 3 of 126 6.2 Spray Drift

................................ ................................ ................................ ........................ 37 7.0 A GGREGATE E XPOSURE /R ISK C HARACTERIZATION

................................ ................................ . 38 8.0 C UMULATIVE E XPOSURE /R ISK C HARACTERIZATION

................................ ............................... 38 9.0 O CCUPATIONAL E XPOSURE /R ISK C HARACTERIZATION

................................ ........................... 38 9.1 Short

- and Intermediate

- Term Handler Risk

................................ ................................ .... 38 9.2 Short

- and Intermediate

- Term Post

- Application Risk

................................ ...................... 40 10.0 R EFERENCES

................................ ................................ ................................ ...................... 41 A PPENDIX A. T OXICOLOGY P ROFILE AND E XECUTIVE S UMMARIES

................................ ............ 42 A.1 Toxicology Data Requirements

................................ ................................ ................... 42 A.2 Toxicity Profiles

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

................................ ............................ 63 A.4 Executive Summaries

................................ ................................ ................................ .. 66 A PPE NDIX B. M ETABOLISM S UMMARY T ABLES

................................ ................................ .......... 117 A PPENDIX C. P HYSICAL

/C HEMICAL P ROPERTIES

................................ ................................ ........ 125 A PPENDIX D. R EVIEW OF H UMAN R ESEARCH

................................ ................................ ............. 126Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 4 of 126 1.0 Executive Summary Dow AgroSciences L LC has petitioned the Agency to register the herbicide halauxifen

- methyl for use on wheat, barley, and triticale and to establish permanent tolera nces for residues resulting from those uses

. This document presents the HED aggregate human health risk assessment for halauxifen

- methyl

. Halauxifen

- methyl ( methyl ( 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- methoxyphenyl)

- 2

- pyridinecarboxylate) is a new arylpicolinate post

- emergence herbicide , and was reviewed as part of a g lobal joint review project

. The review partners we re the United States Environmental Protection Agency ( U. S. EPA ) , the Pest Management Regulatory Agency (PMRA) of Canada and the Austr alian Pesticides and Veterinary Medicines Authority (APVMA)

. The submitted studies make up a complete database for the purpose of conducting an aggregate human health risk assessment. No risks of concern were identified in this assessment, from either oc cupational exposures or from dietary exposures , for the general U.S. population or any population subgroup

. Under the proposed use pattern, h alauxifen

- methyl may be applied as a post

- emergence broadcast spray through a variety of application methods inc luding ground and aerial equipment at a maximum single application rate of 0.0091 lb ai/A. There is a potential for occupational exposure associated with handler activities (i.e., mixing, loading, and applying) and for workers re
- entering treated areas. Based on the use sites, there is the potential for dietary exposure to halauxifen

- methyl and/or its breakdown products via consumption of treated crops and from drinking water

. There are no proposed uses that would result in residential exposures

. The toxicology database for halauxifen

- methyl is considered adequate and no additional data are required at this time. Following oral exposure, halauxifen

- methyl is rapidly hydrolyzed in the liver to its major metabolite, XDE

- 729 acid, which then enter s s ystemic circulation. The liver is therefore exposed pre

- systemically to parent halauxifen

- methyl prior to its being metabolized to XDE

- 729 acid, but other organs are exposed to XDE

- 729 acid. The guideline studies in support of registration of halauxifen

- methyl were conducted on XDE

- 729 acid and identified the kidney as the main target organ. Although halauxifen

- methyl is rapidly converted to XDE

- 729 acid, kidney effects were not observed after halauxifen

- methyl exposure because the blood levels of the ac id from exposure to the parent compound were lower than those seen with comparable exposure to the acid per se
. Bridging studies designed to compare the toxicity profile of halauxifen

- methyl and XDE

- 729 acid identified liver as the target organ for halaux ifen

- methy l

. However , chronic toxicity data from the XDE

- 729 acid could not be used for bridging because liver toxicity from subchronic exposure to halauxifen

- methyl occurred at lower doses. In lieu of conducting long

- term studies on halauxifen

- methyl, m echanistic studies were conducted. These studies identified activation of the liver aryl hydrocarbon receptor (AhR) as the mode of action (MOA), and the molecular initiating event (MIE), for liver toxicity, for which increased liver Cyp1a1 gene expression serves as a biomarker. Due to the distinct toxicities of the halauxifen

methyl and XDE

- 729 acid and the unique MOA for liver toxicity of halauxifen

- methyl, risk from the two compounds was assessed separately. Therefore, bridging and mechanistic studies were considered , along with the guideline studies, in selection of the dose and endpoint for halauxifen

methyl. Based on the abundance of guideline and mechanistic data available, a MOA approach was used for the identification and characterization of haza rd. A point of departure (POD) forHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 5 of 126 increased Cyp1a1 expression was identified in the rat 90

- day dietary study and was selected for risk assessment of halauxifen

- methyl , since it protects for the initial step in liver toxicity.
There was no evidence of dermal toxicity, neurotoxicity or immunotoxicity. I nhalation studies were waived because test atmospheres could not be generated for either compound, and the margin of exposure ( MOEs ) for inhalation exposure using the POD of 3 mg/kg/day were adequately pr otective (>1000) ; therefore, an additional database uncertainty factor (UF DB ) is not required in the absence of the inhalation study

. Neither compound was acutely toxic (Category IV). Both are mild eye irritants (Category III) but not dermal irritan ts or sensitiz ers
. XDE

- 729 acid is classified as “not likely to be carcinogenic to humans.” Halauxifen

- methyl is classified as “not likely to be carcinogenic to humans at doses that do not induce Cyp1a1 expression , ” based on the premise that AhR activation and subsequent promotion of hepatocellular tumors due to a sustained increase in hepatocellular proliferation depend upon this MIE. Neither show evidence of genotoxicity. There was no evidence of increased prenatal susceptibility to either compound and no evidence of postnatal susceptibility to XDE

- 729 acid. Residual c oncerns for postnatal susceptibility to halauxifen

- methyl in the absence of reproductive toxicity data are low, due to selection of a highly conservative endpoint and assumptions for dieta ry exposure , and the low level of exposure expected from proposed use patterns. T he FQPA safety factor (SF) is therefore reduced to 1x.
A cute dietary assessment s w ere not conducted due to lack of single

- dose effects. For chronic dieta ry exposure to XDE

- 729 acid, the NOAEL of 20.3 mg/kg/day from the rat chronic toxicity/ carcinogenicity study was selected, based on kidney effects. For chronic dietary ex posure to halauxifen

- methyl, the POD of 3.0 mg/kg/day for mildly increased liver Cyp1a1 expression in t he rat 90

- day study was selected

. Although this endpoint was not adverse

- no liver effects were observed at the POD

- in the absence of long

- term data, it is considered protective because liver toxicity will not result without the MIE. For non

- dietary e xposure, only occupational endpoints for halauxifen

- methyl were selected because t here are no proposed residential uses

. O ccupational handlers /applicators will be exposed only to the parent in product formulations. Dermal exposure was not assessed due to lack of toxic effects via the dermal route . For inhalation exposure, the MIE ( POD of 3 mg/kg/day ), selected in the absence of inhalation data, was considered conservative and protective of potential portal of entry and systemic effects. Toxicity via the i nhalation route was assumed by default to be equivalent to toxicity via the oral route

. The plant and livestock metabolism studies are adequate for determination of the residues of concern in plants and livestock f or both tolerance enforcement and risk assessment for the purposes of the current tolerance petition

. HED has determined that the residue of concern in primary crops and rotational crops for both tolerance enforcement and risk assessment is parent halauxi fen

- methyl only. The residue of concern in ruminants and poultry for both tolerance enforcement and risk assessment is the metabolite X11449757 ( 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- hydroxyphenyl)

- pyridine

- 2

- carboxylic acid )

. Based on environmental fa te studies, the breakdown of halauxifen

- methyl in drinking water is more complex. In drinking water, the residues may include parent halauxifen

- methyl, as well as transformation products XDE

- 729Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 6 of 126 acid, X11449757, X11406790, degradates 1, 2, 4, 10, 11, and 14. The distribution of residues between these compounds will be dependent on the source of the drinking water (surface or groundwater) as well as the amount of elapsed time since application of the product. Regulatory enforcement in primary crops is bas ed on parent halauxifen

- methyl only. Enforcement of tolerances can be accomplished by a validated LC

- MS/MS analytical method with a limit of quantitation of 0.01 ppm. No livestock commodity tolerances are required for the proposed uses because there is n o reasonable expectation of finite residues in these commodities

. Following application of halauxifen

- methyl according to the directions for use on the proposed label s , residues of halauxifen

- methyl are expected to be below the limit of quantitation of 0.01 ppm in wheat and barley grain and most of the associated livestock commodities ; the only detectable residues were found in wheat straw, hay, and forage

. As discussed above, the toxicological effects caused by parent halauxifen

- methyl and the XDE

729 acid metabolite are different. The parent targets the liver, while the metabolite causes kidney effects. In the field trials, no residues of parent or acid were found in wheat or barley grain. The acid metabolite is a major degradate in drinking water. As the parent and metabolite have different toxicological effects, separate chronic toxicity endpoints were selected for them and separate c hronic dietary exposure assessments were performed for the two compounds. For parent halauxifen

- methyl, an assessment was performed for food and drinking water. Th is assessment was based on tolerance level residues in food and the assumption that 100% of all barley, wheat, and triticale consumed will be treated with halauxifen

- methyl. Drinking water residues for halauxifen

- methyl were negligible and did not contribute to the dietary exposure. For the acid metabolite, the assessment was based on drinking water alone because the acid metabolite is not a residue of concern in food, but it is a residue of concer n in water. Dietary risk estimates for halauxifen

- methyl and XDE

- 729 acid are well below HED’s level of concern
. In both of the chronic dietary exposure assessments that were performed, the general U.S. population and all population subgroups used < 1 % of the chronic population adjusted dose (cPAD). For the chronic dietary exposure assessments with parent and XDE

- 729 acid , the maximum exposure estimates were for the “all infants” subpopulation

. As there are no residential uses for halauxifen

- methyl , there are no residential exposure contributions to the aggregate risk estimates

. Therefore, aggregate risk estimates are equivalent to the dietary risk estimates and are also below HED’s level of concern. For occupational scenarios, long

- term exposures ( > 6 months) are not expected to occur; however, short

- (1

- 30 days) and intermediate

- term (1

- 6 months) exposures may occur. Occupational assessments for halauxifen

- methyl are based on inhalation exposures only

. All occupational handler scenarios for the p roposed uses resulted in MOEs greater than 100 at baseline personal protective equipment ( PPE; i.e., baseline attire with no respirator; engineering control for aerial applications (enclosed cockpits)) and, therefore , are not of concern . Although a POD fr om an oral study was used to assess handler inhalation risk, the calculated MOEs are all ≥2,500, thus providing an ample margin to account for any uncertainties in the route

- to

- route extrapolation. Based on the Agency's current practices, a quantitative n on
- cancer occupational post

- application inhalation exposure assessment is not being performed for halauxifen

- methyl atHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 7 of 126 this time. If new policies or procedures are put into place, the Agency may revisit the need for a quantitative occupational post

- applic ation inhalation exposure assessment for halauxifen

- methyl

. There are no human health risk concerns that would preclude granting the requested registrations for halauxifen

- methyl and establishing tolerances for residues at the recommended levels

. Potential areas of environmental justice concerns were considered in this human health risk assessment to the extent possible

. For more information, see Section 3.5. This risk assessment relies in part on data from studies in which adult human subjects w ere intentionally exposed to a pesticide or other chemical

. Those studies are subject to ethics review, have received that review, and are compliant with applicable ethics requirements.
2.0 HED Recommendations Provided the recommended changes are made t o the halauxifen

- methyl end

- use product label s (see Section 2.3), HED recommends registering halauxifen

- methyl for use on wheat, barley, and triticale

. Furthermore, HED recommends establishing permanent tolerances for residues related to halauxifen

- methyl as discussed in Section 2.2.
2.1 Data Deficiencies/Conditions of Registration
None.
2.2 Tolerance Considerations
2.2.1 Enforcement Analytical Method Suitable methods for tolerance enforcement have been developed and independently validated. For all matrices and analytes, t he limit of quantitation ( LOQ ) , defined as the lowest spiking level where acceptable precision and accuracy data were obtained, was determined to be 0.01 mg/kg. The limit of detection (LOD) was defined to be 30% of the LOQ (i.e., 0.003 mg/kg).
The FDA multi

- residue methods are not suitable for detection and enforcement of halauxifen

methyl and XDE

- 729 acid in non

- fatty matrices. T he QuEChERS multiresidue method was evaluated for the quantitative determination of residues of halauxifen

- methyl and XDE

- 729 acid in agricultural commodi ties representative of the four OECD crop types: wet crop, dry crop, oily crop, and acidic crop

. This QuEChERS multiresidue method was found to be adequate for the determination of residues of halauxifen

- methyl and XDE

- 729 acid in the four types of crops tested. Plant Commodity Enforcement Method
Residues of halauxifen

- methyl and XDE

- 729 acid are extracted with acetonitrile/water (80:20, v/v) and the sample is centrifuged. An aliquot of supernatant is filtered/centrifuged and cleaned up by solid

- phas e extraction. After preparation, the samples a re analyzed by liquid chromatography using a C8 column coupled with positive

- ion electrospray LC

- MS/MS,Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 8 of 126 monitoring two MS/MS transitions characteristic of each analyte and one MS/MS transition characteristic o f each internal standard. For halauxifen

- methyl, t he ion transition m/z 345 [M+H] + → 250 is used for quantification, and the transition m/z 345 → 235 is used for confirmation. For XDE

- 729 acid, the ion transition m/z 331 [M+H] + → 250 is used for quantification, and the transition m/z 331 → 285 is used for confirmation. Livestock Commodity Enforcement Method Dow AgroSciences proposed the m ethod described in Report 110505 as the analytical enforcement method for halauxifen

- methy l and XDE

- 729 acid in livestock matrices. However, HED has determined that the residue of concern in livestock commodities is the metabolite X11449757. Based on the available data for residues in livestock feedstuffs associated with this petition, quanti fiable residues in livestock matrices are not expected, and a validated livestock enforcement method is not necessary at this time. In the cattle feeding study, samples of cattle commodities were analyzed using a modification of the proposed enforcement m ethod. In the event that tolerances are needed for livestock commodities in the future, the petitioner will need to submit an adequate enforcement method for residues of X11449757 in livestock commodities and an independent laboratory validation ( ILV ) for the method.
2.2.2 International Harmonization
Halauxifen

- methyl is a new active ingredient, and no MRLs have been established by Codex, Canada, or Mexico. Th e U.S., Canada, and Australia , all agreed to use the OECD MRL calculat ion procedures for this tolerance petition. In addition, the three countries are in agreement with respect to the residues of concern for tolerance enforcement. The residue of concern in plant commodities is parent halauxifen

- methyl only. The residue of concern in liv estock commodities is the metabolite X11449757.
The tolerances/ MRLs that are being recommended by the U.S. and Canada are harmonized to the extent possible. In the cases where tolerances/ MRLs are not being harmonized, it is because of policy differences in the way the two countries establish tolerances/ MRLs. The only MRLs that are not being harmonized are those for livestock feed items and livestock commodities. Canada does not establish MRLs for livestock feed items, whereas the U.S. does establish tol erances
. The U.S. is establishing tolerances for residues in wheat forage, hay, and straw , as well as for barley hay and straw. On the other hand, the U.S. generally does not establish tolerances for livestock commodities when there is no reasonable expe ctation of finite residues. Canada establishes LOQ MRL s in these situations. As a result, Canada is establishing LOQ MRL s for ruminant, poultry, and swine commodities, whereas the U.S. is not establishing tolerances

. 2.2.3 Recommended Tolerances
HED re commends that 40 CFR be amended by establishing tolerances for the plant commodities listed in the table below. T he residue definitions for plant and livestock commodities are different, as a result, HED has prepared two separate tolerance summary tables for them. For plant commodities, the tolerances should be established as follows: Tolerances are established for residues of halauxifen

- methyl, including its metabolitesHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 9 of 126 and degradates, in or on the commodities in the table below. Compliance with the tolerance levels specified below is to be determined by measuring only halauxifen

- methyl (methyl (4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- methoxyphenyl)

- 2

- pyridinecarboxylate).

Table 2.2.3.1
. Tolerance Summary for Halauxifen
- methyl
. Commodity as Proposed by Petitioner Proposed Tolerance (ppm) Recom

mended Tolerance (ppm) Comments

(correct commodity definition) Plant Commodity Tolerances barley, grain 0.01 0.01 Barley, grain barley, hay 0.01 0.01 Barley, hay barley, straw 0.01 0.01 Barley, straw wheat, forage 0.5 0.50 Wheat, forage wheat, grain 0.01 0.01 Wheat, grain wheat, hay 0.04 0.03 Wheat, hay wheat, straw 0.015 0.015 Wheat, straw Dow AgroSciences did not propose a tolerance for triticale, and does not need to because a tolerance for wheat automatically applies to triticale (40 CFR §180.1(g)). Tolerances are not needed for livestock commodities due to the lack of quantifiable residues in livestock commo dities associated with the proposed uses . The tolerances that the petitioner proposed are listed in the following table

.
Table 2.2.3.2
. Petition Proposed Livestock Commodity Tolerance Summary for Halauxifen
- methyl
.

Commodity Proposed Tolerance (ppm) Recommended

Tolerance (ppm) Comments

(correct commodity definition) cattle, fat 0.01 None cattle, meat 0.01 None cattle, meat byproducts 0.01 None goat, fat 0.01 None goat, meat 0.01 None goat, meat byproducts 0.01 None horse, fat 0.01 None horse, meat 0.01 None horse, meat byproducts 0.01 None milk 0.01 None sheep, fat 0.01 None sheep, meat 0.01 None sheep, meat byproducts 0.01 None
2.2.4 Revisions to Petitioned
- For TolerancesHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 10 of 126 As can be seen in Table 2.2.3.1 , above, the only recommended tolerance for treated crops that is different from the proposed tolerance is that for wheat hay. The petitioner proposed a tolerance of 0.04 ppm, whereas HED determined that a tolerance of 0.03 ppm is appropriate. When the petitioner determined the proposed tolerances, it included the metabolite XDE

- 729 acid as a residue of concern. HED subsequently determined that this metabolite is not a residue of concern for tolerance enforcement. The petitioner should submit a revised Section F in which a tolerance of 0.03 ppm is proposed for wheat, hay. In addition, the petitioner proposed livestock commodity tolerances, but HED has determined that they are not required because quantifiable residues are not expected in livestock commodities

. 2.3 Label Recommendations The proposed labels specify the single application rates and the maximum seasonal application rate s. HEDs risk assessment is based on these maximum application rates. It is noted that the labels do not specify the maximum number of applications that may be made per season

. For this assessment, HED has inferred the number of applications per season by comparing the single application rate to the maximum seasonal application rate . On each of the labels, the petitioner needs to specify the maximum number of applications that may be made per growing season. Some of the end

- use products proposed for registration contain multiple active ingredients. For those products, RD should ensure that the use restrictions on the proposed labels comply with any restrictions associated with the co

- formulated active ingredients.
3.0 Introduction
3.1 Chemical Identity

Table 3
. 1
. Nomenclature and Identification Information for Halauxifen
- Methyl
. Active Ingredient

Common Name Halauxifen

- methyl Company Experimental Name XDE

- 729 methyl or XDE

- 729 methyl ester Company Experimental Number X11393728 IUPAC Name Methyl 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- methoxyphenyl)pyridine

- 2

- carboxylate Molecular Formula C 14 H 11 O 3 N 2 Cl 2 F Molecular Weight 345.16 CAS Name M ethyl 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- methoxyphenyl)

- 2

- pyridinecarboxylate CAS Number 943831

- 98

- 9Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 11 of 126 3.2 Physical/Chemical Characteristics
Technical

- grade halauxifen

- methyl is a solid at room temperature. The compound has a low vapor pressure ( 1.1 x 10 - 10 mm Hg at 25°C), and a low proposed use rate (0.0091 lb ai/A) ; therefore, there is a relatively low likelihood of exposure to halauxifen

- methyl in the vapor phase

. The technical

- grade material has a log K OW of 3.8 indicating a somewhat

- fat

- soluble compound, but not to the degree that bioaccumulation is of particular concern

. A listing of the physical/chemical properties of halauxifen

- methyl and the XDE

- 729 acid metabolite are provided in Appendix C.
3.3 Pesticide Use Pattern
Halauxifen

- methyl has been proposed for use on wheat, barley, and triticale. Halauxifen

- m ethyl is a post

- emergent herbicide developed for control of a broad spectrum of broadleaf and sedge weeds

. Application is intended prior to harvesting crops in the f ield. The proposed use directions are summarized in Table 3.3. Four agricultural products are proposed for use on wheat, barley, and triticale: GF

- 2685, GF

- 2686, GF

- 2687, and GF

- 2688. These end use products may be applied through a variety of applicat ion methods including: aerial and ground equipment. These products are not to be applied using any type of chemigation equipment. The proposed Restricted Entry Interval (REI) is 12 hours for all crops. The labels do not specify the maximum number of applications that may be made. The recommended single application rate is approximately 0.005 lb ai/A. For GF

- 2685, the maximum seasonal application rate is 0.0091 lb ai/A. These application rates indicate that the total number of applications of GF

- 268 5 should not exceed two.
The label for GF

- 2685 states that livestock may be grazed 1 day following application. The only active ingredient in this product is halauxifen

- methyl. The labels for GF

- 2686, GF

- 2687, and GF

- 2688 state that livestock may be gra zed 7 days following application. These products contain either florasulam or fluroxypyr in addition to halauxifen

- methyl. The PPE statement on the proposed labels requires applicators and other handlers to wear: long

sleeved shirt and long pants, chem ical

- res istant gloves, and shoes plus socks.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 12 of 126 Table 3.3
. Proposed Uses of Halauxifen
- Methyl on Wheat, Barley, and Triticale
.
Applic. Timing, Type, and Equip. a Formulation (EPA

Reg. No.) b Application Rate (lb ai/A) c Max No.

Applic. Per Season d Max. Seasonal

Applic. Rate ( lb ai/A ) PHI e Use

Directions and Limita
tions f

Ground and Aerial Equipment GF

- 2685 WG

(62719

- AA N ) 0.004 5

- 0.0091 1

- 2 (depending on rate) 0.00 91

60 Days See Footnotes GF

- 2686 WG (62719

- AA R ) 0.0049 1 0.0049
GF

- 2687 WG (62719

- AA E ) 0.0049 1 0.0049
GF

- 2688 EC (62719

- AAG) 0.0058 1 0.0058 a: Do not apply using any type of chemigation equipment

. Do not apply halauxifen

- methyl to more than two growing seasons per year (i.e., Spring and/or Fall)

. b : WG: Water dispersible granule

. EC: Emulsifiable concentrate

. c : For the proposed label GF

- 2685, the maximum single application rate of 0.0091 lb ai/A is for heavy infestations or larger stages of weeds. d: The maximum number of applications per season per year is not specified on the label s . Those given in the table are inferred based on the single application rates and the maximum seasonal application rate. e : PHI: Do not apply within 60 days of crop harvest. Do not apply closer than 21 days before cutting of hay. Livestock may be grazed on treated crops 1 day following application. f : To minimize spray drift, apply in a total spray volume of 3 gallons or more per acre.
3.4 Anticipated Exposure Pathways Based on the proposed use pattern on grain cr ops, h umans may be exposed to halauxifen

- methyl

related residues in food from treated crops and from migration of residues to surface and/or groundwater sources of drinking water

. Fro m crops, the exposure will be primarily to parent halauxifen

- methyl

. In drinking water, the residues may include parent halauxifen

- methyl, as well as transformation products XDE

- 729 acid, X11449757, X11406790, and degradates 1, 2, 4, 10, 11, and 14. The distribution of residues between these compounds will be dependent on the source of the drinking water (surface or groundwater) as well as the amount of elapsed time since application of the product. At this time, there are no proposed residential uses of halauxifen

- methyl , and exposure via residential pathways is not expec ted. In an occupational setting, t here is a potential for occupational exposure associated with handler activities (i.e., mixing, loading, and applying). Post

- application exposure is expected for workers re
- entering treated areas.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 13 of 126 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 M inority Populations and Low

- Income Populations," ( http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf

. As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups according to well

- established procedures

. In line with OPP policy, HED estimates risks to population subgroups from pesticide exposures that are based on patterns of that subgroup’s food and water consumption, and activities in and a round 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 as sessments for all registered food uses of a pesticide

. These data are analyzed and categorized by subgroups based on age and ethnic group

. Additionally, OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments are performed when conditions or circumstances warrant

. Whenever appropriate, non

- dietary exposures based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or playing on treated areas p ost

- application are evaluated

. Further considerations are currently in development as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditional dietary patterns among specific subgroups.
4.0 Hazard Characterization and Dose
- Response Assessment
Th e hazard characterization for halauxifen

- methyl takes into consideration the distinct toxic ological profiles of both halauxifen

- methyl and its major metabolite, XDE

- 729 acid. Following oral exposure, halauxifen

- methyl is absorbed in the gut and transported via the enterohepatic circulation to the liver, where it is rapidly and completely metabolized via hydrolys is at the methyl ester to form XDE

- 729 acid , before entering systemic circulation. Therefore, the liver is exposed pre

- systemically to halauxifen

- methyl, but exposure to other tissues and organs through systemic circulation is to XDE

- 729 acid. Based on t he rapid hydrolysis of the parent to acid and the similar ity of their structures, it was anticipated that they would show comparable toxicity. The g uideline toxicology studies for halauxifen

- methyl were conducted on XDE

- 729 acid, with additional bridging studies conducted on halauxifen

- methyl (with the exception that the immunotoxicity study was conducted on the parent compound)

. In fact, very different toxicological profiles were observed. Comparison of the subchronic rat studies showed that the kidne y is the target organ for XDE

- 729 acid, whereas for halauxifen

methyl , the target organ is the liver

. Furthermore, the liver effects of halauxifen

- methyl occur red at significantly lower doses than those at which XDE

- 729 acid caused kidney toxicity

. Therefore, the data on halauxifen

- methyl cannot be used to bridge to the acid.
In lieu of conducting a complete set of guideline toxicity studies on halauxifen

- methyl to evaluate long

- term toxicity , mechanistic studies were performed to characterize the mode of action (MOA) for liver toxicity , with the anticip ation that this would provide adequate information, when considered together with the bridging data and studies on XDE

- 279 acid , toHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 14 of 126 characterize toxicity from halauxifen

- methyl

. The mechanistic data support a MOA of activation of liver aryl hydrocarbon receptor (AhR) by halauxifen

- methyl (but not XDE

- 729 acid) . AhR activation is a well

- known MOA that has been characterized for a number of compounds, most notably dioxin, and that may affect diffe rent tissue types ( reviewed in Nebert et al. , 2004; Cohen, 2010; Safe et al., 2013 ). In the liver, this MOA causes liver hypertrophy , histopathology and cellular proliferation, and with sustained AhR activation, over time may promote the formation of hepa tocellular tumors due to increased liver cell proliferation . AhR activation through binding by the ligand, halauxifen

- methyl, is considered the molecular initiating event (MIE) , an initial prerequisite event upon which the subsequent effects are dependent . An early indicator of AhR activation is increased expression of the liver Cyp1a1 gene, which serves as a readily measurable biomarker for the MIE. Therefore, in addition to the liver toxicity reported in the rat subchronic toxicity studies, increases i n Cyp1a1 gene expression may be used to identify dose levels at which there is the potential for liver effects from AhR activation with long

- term exposure , in the absence of long

- term animal toxicity studies

. The MOA and supporting data are summarized bel ow ( Section 4.3.2 ) and in greater detail in a separate document (TXR # 00 51197 ). The extensive guideline and mechanistic study database for halauxifen

- methyl and XDE

- 729 acid allowed the Agency to use a MOA approach for characterization of hazard and identification of a MIE for halauxifen

- methyl and endpoints for risk assessment

. 4.1 Toxicology Studies Available for Analysis The toxicology database is considered adequate for assessment of human health risk from exposure to halauxifen

- methyl. No additional studies are required at this time

. T his determination is in accordance with th e g lobal r eview p artners, Australia OCS and Canada PMRA, as well as the European Union. In mammals, halauxifen

- methyl is rapidly hydrolyzed (demethylated) in vivo to its major metabolite, XDE

- 729 acid, and therefore the majority of the guideline toxicity studies were conducted on the acid, with bridging studies conducted on halauxifen

- methyl

. The following guideline studies were submitted:

Halauxifen
- methyl XDE
- 729 acid Acute toxicity battery (except for acute

inhalation) Acute toxicity battery (except for acute inhalation) 28

- and 90

- day subchronic (rat) 28

- and 90

- day subchronic (rat, mouse, dog)

- 28

- day dermal (rat) Prenatal developmental (rat, rabbit) Prenatal developmental (rat, rabbit)

- Two

- generation reproductive toxicity (rat)

- Chronic toxicity (dog)

- Carcinogenicity (mouse)

- Chronic toxicity/carcinogenicity (rat) Genotoxicity battery Genotoxicity battery

- Neurotoxicity (rat, acute and subchronic) Metabolism (rat) Metabolism (rat) Dermal penetration, rat ( in vivo and in vitro )

-

Immunotoxicity (rat)

- A 28

- or 90

- day inhalation study was not conducted for either compound because of difficulties in generating a test atmosphere containing respirable particles (1 to 4 μm diameter) and clogging of the aerosol delivery appa ratus. However, the available oral endpoint was adequatelyHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 15 of 1 26 protective for inhalation exposure, based on high MOEs ( Level of Concern or LOC=1000; MOEs all 2500 or greater ), and both compounds show low volatility. Based on these considerations, the HED Haza rd Science Policy Council (HASPOC) concluded that t he inhalation study is not needed (TXR No. 0056954). The bridging studies on halauxifen

- methyl indicated that i t s toxicological profile is very different from that of XDE

- 729 acid. Liver toxicity, an e ffect not observed with the acid form, was observed at lower dose levels of halauxifen

- methyl than those of XDE

- 729 acid at which kidney effects were observed

. To elucidate the basis for these differences, m echanistic studies on halauxifen

- methyl were als o submitted to characterize the mode of action (MOA) for liver toxicity, and to evaluate the comparative metabolism of XDE

- 729 acid in the dog, rat and mouse. Genotoxicity studies were submitted for the minor metabolite X11449757 [ 4

- Amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- hydroxyphenyl)

- pyridine

- 2

- carboxylic acid ] , which is derived from XDE

729 acid by demethylation at the methoxy group of the phenyl ring

. No additional data are required for this metabolite, as it is a more polar compound and is not expec ted to be more toxic than either halauxifen

- methyl or XDE

- 729 acid. There is no published literature information specific to halauxifen

- methyl available for consideration, as this is a new proposed active ingredient.
4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)
Rat metabolism studies were conducted on both XDE

- 729 acid and halauxifen

- methyl. XDE

- 729 acid wa s rapidly and well

- absorbed following a single 10 mg/kg gavage dose , with a time to peak plasma concentration ( T max ) of ~ 0.2 hr. Elimination wa s primarily via the urine (68

- 92% administered dose (AD)), with 11

- 29% excreted in feces and most of the AD excreted within 24 hr of dosing

. Fecal excretion was mostly due to biliary excretion, based on fecal excretion in the IV dose gr oup. Females excreted somewhat higher radioactivity levels in the urine than males. Elimination was biphasic, with a n initial phase of 0.5

- 0.9 hr and a slower terminal phase of 6

- 16 h at 10 mg/kg. At 750 mg/kg, the initial elimination phase was slower ( 2.7

- 4.3 hr), but the terminal phase was faster ( 5.6

- 8.7 hr ), and the p lasma area under the curve ( AUC ) was 2.0

- 2.6

- fold higher than the low dose

. Taken together, the data suggest saturation of excretion at higher doses. Radioactivity was not retained in tissues. XDE

- 729 acid was mostly excreted unmetabolized. Minor metabolites (<5% AD) included the acyl

- glucuronide conjugate , hydroxy XDE and O

- demethylated XDE

- 729 acid , and corresponding sulfate and glucuronide conjugates.
Halauxifen

- methyl showed si milar kinetics following a 10 mg/kg gavage dose. T max was slightly longer (0.50

- 0.44 h, M/F) than XDE

- 729 acid (0.19

- 0.15 hr, M/F), and C max was lower ( 1.6 to 3.6

- fold lower ), but maximum absorption and biphasic elimination t 1/2 s, and the AUCs, were compa rable. Radioactivity was not retained in tissues. A t or near the peak plasma radioactivity concentration , the primary analyte was XDE

- 729 acid, indicating rapid hydrolysis of the ester in vivo

. The major radioactive component in urine and feces was XDE

- 729 acid (80% AD), with minor metabolites (<5% AD) similar to those of XDE

- 729 acid

.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 16 of 126 Additional metabolism studies were conducted to evaluate metabolism of XDE

- 729 acid in the Beagle dog and comparative metabolism in the rat and mouse. D ogs also sh owed rapid absorption and excretion, primarily in the urine, with most radioactiv ity excreted unmetabolized. C learance was somewhat slower than in the rat. In the comparative study, absorption, metabolism and elimination were similar in rats and mi ce at the doses tested

. Toxicokinetic evaluation s were conducted in many of the oral toxicity studies on halaux ifen

methyl and XDE

- 729 acid , and included assess ment of parent and/or metabolite in the blood and, in some studies, the urine and liver

. In rat 28

- and 90

- day dietary studies on halauxifen

- methyl, XDE

- 729 acid was found in the blood at all dose levels; low levels of halauxifen

- methyl were observed only at the highest doses. Urinary excretion was primarily as XDE

- 729 acid, with very low levels of halauxifen

- methyl at or above the study LOAELs. Blood levels of XDE

- 729 acid in rats administered halauxifen

- methyl were below the levels observed at comparable dietary doses of XDE

- 729 acid, consistent with the lack of observed kidney toxicity in studie s on halauxifen

- methyl and rapid excretion of the acid. The most abundant analyte in liver in the rat 90

- day study on halauxifen

- methyl was XDE

- 729 acid, but halauxifen

- methyl was detected in all treated groups and showed dose linearity. In males, but no t females, XDE

- 729 acid as percent AD decreased at the highest dose. In rat and rabbit developmental studies on halauxifen

- methyl, only XDE

- 729 acid was observed in maternal and fetal blood. Levels in fetal blood were lower than maternal blood in the rat (about 65% of maternal levels) and rabbit (5

– 27% of maternal levels). Toxicokinetic data from studies on XDE

- 729 acid showed linearity of the AUC except at high doses.

4.2.1 Dermal Absorption Dermal absorption of halauxifen

- methyl following a 10 hr exposure was evaluated in a rat in vivo dermal absorption study and a comparative in vitro dermal absorption study using rat and human skin samples. Both studies were conducted using a liquid concentrate formulation, GF

2573, containing [ 14 C]

- halauxifen

- m ethyl and applied undiluted or as two field dilutions, at concentrations of 75, 1.5 or 0.25 μg ai/cm 2 , respectively. Absorption was monitored in the in vivo study up to 144 hr (concentrate) or 192 hr (dilutions) and in the in vitro study, up to 24 hr.
In vivo percent dermal a bsorption (absorbed plus absorbable dose, excluding the two outermost layers of skin) was 1 3, 15 and 24% of the applied dose (highest to lowest concentration , respectively ). Toxicokinetic evaluations were performed in the in vivo rat study and show that following absorption, internal exposure is to XDE

- 729 acid. Blood levels of radioactivity at 4 and 8 hr post

- application showed that only XDE

- 729 acid was present, indicating rapid hydrolysis of the absorbed parent compound to the acid metabolite following dermal absorption. This is consistent with the findings of a comparative in vitro tissue S9 methyl ester hydrolysis study in which halauxifen

- methyl was hydrolyzed to XDE

- 729 acid in the blood, although activity was lower than in the liver. Hydrolysis activity of the skin tissue itself was not examined.
The ratio s of in vitro dermal abs orption in human:rat skin samples were 0.18, 0.37 and 0.60 (highest to lowest dilution, respectively)

.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 17 of 126 Based on t he in vivo der mal absorption study, the data indicate that up to 24% of the applied dose was absorbed in the rat following a 10 hr exposure to halauxifen

- methyl. However, given that no toxicity was observed in the rat 28

- day dermal toxicity study, a dermal assessment is not required.

4.3 Toxicological Effects
Halauxifen

- methyl is rapidly hydrolyzed in vivo at the methyl ester to its major metabolite, XDE

- 729 acid

. The toxicity profiles of the two compounds in oral toxicity studies showed significant differences, because of the rapid demethylation of the parent compound to the acid

. The major target organ of halauxifen

- methyl following oral exposure is the liver, which results from direct, pre

- systemic exposure of the liver from the enterohepatic circulation . In subchronic (28

- and 90

- day) dietary studies in the rat, increased liver weight, hepatocellular hypertrophy, increased hepatocyte mitotic figures, vacuolization consistent with fatty change and increased cholesterol levels were reported at the same do se levels in both studies. These findings were minimal in both studies, suggesting lack of progression in exposures of short

- to intermediate

duration

. The liver effects were associated with significant increases (≥1500

- fold above controls) in the expr ession of the cytochrome enzyme Cyp1a1. Mechanistic studies on halauxifen

- methyl identified activation of the aryl hydrocarbon receptor (AhR) as the molecular initiating event (MIE) in the mode of action (MOA) leading to hepatic toxicity

. The mechanistic data are summarized in greater detail below in Section 4.3.2 , and additional details are provided in a separate document (TXR # 0051197)

. The identification of liver AhR activation as the MOA for liver toxicity is relevant to both systemic and carcinogeni c effects from long

- term exposure, as described below. Thyroid follicular cell hypertrophy was also observed at doses causing liver effects. Increased expression of liver UGT enzymes, measured in the rat 90

- day oral study, suggests that the thyroid eff ects are secondary to increased liver metabolism of thyroid hormones. Other reported effects included decreased body weight/weight gain, and thymic atrophy with decreased thymus weight. Hyperplasia of erythroid cells in the bone marrow and slight increas es in extramedullary hematopoiesis in the spleen were reported in the 28

- day rat study. However, effects on the thymus and the hematopoietic system were only observed at high dose levels that were significantly greater than the doses showing liver or thyr oid effects.
In contrast , following oral exposure to XDE

- 729 acid , the kidney is the major target organ. Due to the rapid hydrolysis of halauxifen

- methyl in the liver following absorption , systemic exposure is to XDE

- 729 acid rather than the parent compo und. Kidney e ffects were observed following exposures of all durations (28

- and 90

- day subchronic and 1

- to 2

- year chronic oral studies) and included increased renal tubule degeneration/regeneration in all species tested. In the rat, increased renal mito tic figures, necrosis, hypertrophy and/or vacuolization of collecting duct epithelial cells, and increased urinary volume were seen in the subchronic studies. Urinary bladder histopathology was observed in female mice after 90 days’ exposure. Increased k idney weights were seen in maternal animals in the rat developmental toxicity study, butHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 18 of 126 histopathology was not performed. The severity of the kidney and urinary tract effects increased with exposure duration in all species. In the mouse carcinogenicity and rat chronic toxicity/carcinogenicity studies, males showed increased mortality secondary to renal and urinary bladder lesions. In the dog chronic study, multifocal glomerulosclerosis in both sexes and in males, granulomas and fibrosis in the renal cor tex, were observed. Following oral administration in all species, effects ranging from decreased body weight and/or body weight gain in rats and dogs to hematological changes in all species were also seen at doses eliciting nephrotoxicity. In the mouse 2 8

- day study, focal necrosis and karyomegaly in the liver were observed at approximately the limit dose, but this was not reported in other mouse studies at similar dose levels. Decreased thymus weights were reported in the dog 28

- and 90

- day studies. No toxicity was observed in rats exposed dermally to XDE

- 729 acid for 28 days up to the limit dose. A dermal toxicity study with halauxifen

- methyl was not conducted, but data from the rat in vivo dermal absorption study indicated that it is rapidly hydrolyze d to XDE

- 729 acid following absorption, based on lack of halauxifen

- methyl in the blood following absorption. Despite the rapid conversion of halauxifen

- methyl to XDE

- 729 acid in vivo , there was no evidence of kidney toxicity in the rat following exposu re to halauxifen

- methyl . A comparison of blood levels of XDE

- 729 acid in rats treated with halauxifen

- methyl vs. XDE

- 729 acid for 28 or 90 days showed that levels following halauxifen

- methyl exposure were lower than the levels observed in studies on XDE

- 7 29 acid , even at comparable mg/kg/day dose levels (Tables 4.3.1.1 and 4.3.1.2). Levels in the kidney were not examined, but the blood concentrations of XDE

- 729 acid associated with kidney toxicity in studies on the acid were not achieved in rats exposed to halauxifen

- methyl , consistent with the lack of kidney toxicity observed from exposure to halauxifen

- methyl

.

Table 4.3.1.1. Comparison of Blood Levels of XDE
- 729 Acid Following Exposure to the Rat in 28
- Day Studies on Halauxifen
methyl and XDE
- 729 A cid.

XDE
- 729 Acid 1 M: 734 m/k/d F: 1000 m/k/d MRID 48557825 Halauxifen
- Methyl 2

M: 782 m/k/d F: 782 m/k/d MRID 48557826 XDE
- 729 Acid 1 M: 250 m/k/d F: 250 m/k/d MRID 48557825 Halauxifen
- Methyl 2

M: 261 m/k/d F: 261 m/k/d MRID 48557826 Halauxifen
- Methyl 2 M: 52 m/k/d F: 52 m/k/d MRID 48557826 Time (hrs) 3 XDE

- 729 Acid in Blood (μg/g) Male 0 63.9 15.7 30 6.6 1.3 Male 3 47.6 11.8 24.4 4.6 0.8 Male 11 36.6 8.1 12 3 0.7 Female 0 103.8 4 32.9 7.4 1.6 Female 3 72.8 2.4 16.3 5.8 1.4 Female 11 44.5 0.3 6.5 2.2 0.6
1 NOAEL = 250 m/k/d. LOAEL = 734 m/k/d (boldface) based on kidney toxicity. Original dose for male was 1000 m/k/d but switched to 500 m/k/d on day 11. 2 NOAEL = 10.8 m/k/d. LOAEL = 55.6 m/k/d based on liver toxicity. 3 The first time point (measured at 6 a.m.) is set as “0” and all other times points are in reference to the zero point. Samples were taken on the final day of dosing.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 19 of 126 Table 4.3.1.2. Comparison of Blood Levels of XDE
- 729 Acid Following Exposure to the Rat in 90
- Day Studies on Halauxifen
Methyl and XDE
- 729 Acid.

XDE
- 729 Acid 1 M: 750 m/k/d F: 750 m/k/d MRID 48557828 Halauxifen
- Methyl 2

M: 500 m/k/d F: 500 m/k/d MRID 48557830 XDE
- 729 Acid 1 M: 250 m/k/d F: 250 m/k/d MRID 48557828 Halauxifen
- Methyl 2

M: 261 m/k/d F: 261 m/k/d MRID 4 8557830 Halauxifen
- Methyl 2 M: 52 m/k/d F: 52 m/k/d MRID 48557830 Time (hrs) 3 XDE

- 729 Acid in Blood (μg/g) Male 0 105.7 17.5 29.3 10.4 12.4 Male 3 51.7 8.5 13.3 4.5 1.5 Male 11 30.8 5.1 9.5 2.9 0.6 Female 0 145 19.1 34.1 11.7 2.4 Female 3 67.3 7.6 14.7 5.3 0.8 Female 11 54.3 9.2 7.0 7.8 0.6
1 NOAEL = 252 m/k/d. LOAEL = 758 m/k/d (boldface) based on kidney toxicity. 2 NOAEL = 10 m/k/d. LOAEL = 52.3 m/k/d based on liver toxicity. 3 The first time point (measured at 6 a.m.) is set as “0” and all other times points are in reference to the zero point. The 3 hour time point for the XDE

- 729 methyl study was actually 5 hours. Samples were taken on the final day of dosing.

Halauxifen

- methyl and XDE

- 729 acid did not show evidence of neurotoxicity. S lightly decreased activity at the highest two dose levels in the acute neurotoxicity study was considered to be an equivocal effect, based on the lack of effects on other parameters and the lack of findings in the subchronic study, and was not considered t o demonstrate neurotoxicity. There were no clinical or histopathological indications of neurotoxicity in any of the other studies on the acid form or halauxifen

- methyl, although the latter was not tested in neurobehavioral studies. No immunotoxicity was reported in rats treated with halauxifen

- methyl, although increased liver weight and decreased thymus weight were observed. There was no evidence of increased qualitative or quantitative pre

- and/or postnatal susceptibility for either halauxifen

- methyl or XDE

- 729 acid. In the case of halauxifen

- methyl, no developmental effects were observed at doses causing decreased maternal weight gain and food consumption toxicity in rats; in rabbits, slight developmental effects (decreased fetal body weight and dela yed ossification of the pubis) were seen in the presence of maternal hepatotoxicity. For XDE

- 729 acid, decreased fetal weights and delays in ossification were observed in the developmental rat study at a dose that caused significant maternal toxicity lead ing to moribund sacrifice of some of the dams during the study. No developmental effects were seen in the rabbit at doses causing slightly decreased maternal body weight gain and food consumption. In a two

- generation reproductive toxicity study conducted in the rat on XDE

- 729 acid, there was no evidence of reproductive toxicity , and no toxicity to offspring was observed, although parental animals showed significant renal toxicity and effects on body weight. A reproductive toxicity study was not conducted on halauxifen

- methyl , but as described below (Sections 4.4 and 4.5), the available data allow selection of endpoints that are protective of offspring

. There was no evidence of carcinogenicity in rat and mouse cancer studies on XDE

- 729 acid, or of genotox ic potential for either compound. XDE

- 729 acid is classified as “not likely to be carcinogenic to humans.” Carcinogenicity studies on halauxifen

- methyl were not conducted. Systemic exposure from halauxifen

- methyl is primarily to XDE

- 729 acid and therefore most tissues are not exposed to the parent compound . However, the mechanistic studies showed that pre
- systemic exposure o f the liver to halauxifen

- methyl results in activation of the AhR receptor,Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 20 of 126 an effect that when sustained (e.g., with long

- t erm exposure) may promote an increased incidence of liver tumors due to increased hepatocellular proliferation. The molecular marker for AhR activation, the molecular initiating event (MIE) for liver toxicity, is increased expression of hepatic Cyp1a1 ( se e Section 4.3.2 and TXR # 0051197 ). The chronic dietary endpoint for halauxifen

- methyl is based on the point of departure (POD) for Cyp1a1 induction from a subchronic study. Since increased expression of Cyp1a1 is one of the early key events in the MOA l eading to hepatotoxicity, although in itself it is not an adverse effect, a dose that is protective of this event will be protective of the potential risk for liver cancer with chronic exposure, based on the rapid onset of AhR activation following initiati on of exposure, and the lack of evidence of temporal progression of liver toxicity in the available studies (28

- and 90

- day). The MOA is considered relevant to human health risk assessment, but in vitro transactivation and AhR receptor binding data sugges t that humans are unlikely to be more sensitive than the rat , and furthermore, that halauxifen

- methyl is a relatively weak AhR agonist due to rapid metabolism and excretion (see TXR #0051197 for further details) . Based on a weight

- of

- the

- evidence consider ation, halauxifen

- methyl is classified as “not likely to be carcinogenic to humans” at doses that do not induce liver Cyp1a1 expression.
Both halauxifen

- methyl and XDE

- 729 acid show low toxicity in acute oral and dermal lethality studies (Toxicity Categ ory III or IV) and are not irritants or dermal sensitizers. Acute inhalation toxicity was not examined due to problems generating the test atmosphere.
4.3.2 Mode of Action fo r Liver Toxicity of Halauxifen
- M ethyl As previously noted, t he observation o f liver effects (including increased liver weights, hepatocellular hypertrophy, mitotic figures in hepatocytes, and hepatocellular vacuolization) in rat subchronic dietary studies with halauxifen

- methyl, but not with XDE

- 729 acid, led the registrant to per form in vitro and in vivo mechanistic studies to further understand the MOA. Based on the results of these studies, a MOA was proposed that involves activation of the AhR through the following key events: (1) pre

- systemic liver exposure to halauxifen

- me thyl (following oral exposure); (2) AhR activation by halauxifen

- methyl (relatively weak binding) as the MIE resulting in: (a) liver enzyme induction (Cyp1a1) and (b) liver weight increases, hepatocyte hypertrophy and vacuolization, and: (3) hepatocellu lar proliferation. (4) unlike many AhR activators, halauxifen

- methyl is rapidly metabolized and not persistent in the body; progression of liver toxicity (including carcinogenic potential) from sustained AhR activation is therefore not expected.
The HED ToxSAC reviewed the MOA and determined that it is plausible and supported by the submitted studies ( TXR No. 0056954). This conclusion is consistent with the determinations of the Global Partners for review of halauxifen

- methyl (Canada PMRA, Australia OCS , and the EU). More detailed summaries of the individual mechanistic studies and the MOA framework are provided in the separate document ( TXR # 0051197 ) . Characterization of AhR as a MOAHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 21 of 126 for toxicity and carcinogenicity in the liver and other tissues has been previously described for other compounds ( reviewed in Nebert et al. , 2004; Cohen, 2010; Safe et al., 2013 )

. Table 4.3.2.1, below, briefly summarizes the supporting data for each key event. Table 4.3.2.1. Summary of Key Events and Supporting Data for Liver Toxicity from Oral Exposure to Halauxifen
- Methyl
. Key Event Data Supporting Key Event Presystemic liver exposure to halauxifen
- methyl

Rapid metabolism to XDE

- 729 acid; systemic exposure is to the acid.

Comparison to humans

Reversibility of findings Levels of halauxifen

- methyl in liver : A dose

- related increase in levels of halauxifen

- methyl in the liver was observed in toxicokinetic studies of halauxifen

- methyl in liver cells in rat 28

- and 90

- day dietary studies. A sharp increase was seen after 7 days’ exposure to the rat at a high dose in a 7

- day dietary study

.

Toxicokinetic data show that XDE

- 729 acid but not halauxifen

- methyl w as found in the blood, except for very low levels o f halauxifen

- methyl that were seen at hig her dose levels. Rat metabolism studies show a rapid half

- life of elimination of radioactivity, primarily as XDE

- 729 acid. PBPK modeling based on in vitro metabolism in rat and human blood and liver S9 showed that halauxifen

methyl and XDE

- 729 acid ach ieve steady

- state concentrations soon after exposure, clear rapidly from blood and liver, and are not expected to accumulate. Yes, the f indings were reversible . Levels of halauxifen

- methyl in liver decreased with time following exposure. Metabolism studies showed that excretion was rapid with minimal retention of radioactivity in tissues. Activation of the AhR

Targeted gene expression Binding of halauxifen

- methyl : In vitro binding of halauxifen

- methyl to the AhR was observed. Transactivation assays in mouse and human cell lines with a luciferase reporter vector showed that halauxifen

- methyl was a weak AhR agonist in mouse but not human cells. In a ligand competition binding assay using rat liver cytosol, halauxifen

- methyl competed weakly with the photoaffinity ligand for rat AhR. XDE

- 729 acid did not bind to AhR. Induction of Cyp1a1 expression : Analyses of gene expressio n were performed in 7

- , 28

- and 90

- day dietary studies. Dose

- related increases in levels of C y p1a1 gene expression were observed for halauxifen

methyl but not XDE

- 729 acid. In vitro gene expression assays in rat, mouse and human hepatocytes also showed a dose

- related increase that was most pronounced in rat hepatocytes. Increases correlated with the presence of halauxifen

- methyl in the liver.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 22 of 126 Table 4.3.2.1. Summary of Key Events and Supporting Data for Liver Toxicity from Oral Exposure to Halauxifen
- Methyl
. Key Event Data Supporting Key Event Associated liver histopathological effects related to AhR activation

Reversibility of findings Hepatocellular hypertrophy , vacuolization, increased liver weight, observed in dietary studies on halauxifen

meth yl, but not XDE

- 729 acid. Liver effects were observed at dose levels that caused pronounced increases in Cyp1a1 expression. A mild increase in hepatocyte Cyp1a1 expression was observed at the NOAEL for liver toxicity, but not at the lowest dose tested , which was identified as a POD for increased Cyp1a1 expression.

Yes, the f indings were reversible . In the 28

- day dietary study, Cyp1a1 expression and liver weights/histopathology were comparable to controls following a 28

- day recovery period. Hepatocellular proliferation

Reversibility of findings observed Increased mitotic indices : Very slight increases in mitotic figures in the liver were observed at high doses in the rat 7

- day study. In the rat 28

- day study, a dose

related incr ease was observed (1/5 and 3/5 animals at 52 and 261 mg/kg/day). No mitotic figures were observed in the rat 90

- day study. Increased BrdU incorporation : Slightly increased BrdU incorporation at 261 mg/kg/day in the rat 28

- day study. Not observed at low er dose levels.
Yes , the findings were reversible . No increases in mitotic figures in the 28

- day study following a 28

- day recovery period. BrdU incorporation levels return towards control levels.
Pre

- systemic liver exposure to halauxifen

- methyl was demonstrated in dietary studies on mice and rats. Pre

- systemic exposure is also expected in humans based on the PBPK modeling. Data supportive of halauxifen

- methyl mediated AhR activation included incr eased enzyme expression of AhR marker genes in vivo and transactivation/binding assays in vitro . Observations of liver weight increases and hepatocyte hypertrophy and vacuolization were seen in multiple rodent studies following treatment with halauxifen

- m ethyl but not XDE

- 729 acid. Finally, increased hepatocellular proliferation was demonstrated by slightly increased bromodeoxyuridine ( BrdU ) uptake by hepatocytes following dietary exposure to halauxifen

- methyl in rats, along with a slight increase in mito tic figures at 7 and 28 days, but not at 90 days. At least partial recovery from induced effects was observed. The key events showed temporal and dose

- related concordance. In the subchronic rat toxicity studies, Cyp1a1 expression was increased by 5 2

- fol d at a dose that showed no evidence of hepatotoxicity ( i.e., study NOAEL). In contrast, there was a marked induction of Cyp1a1 (>1500

- fold) at doses where liver effects were also observed ; however , liver toxicity at the LOAEL was minimal and did not becom e more severe, or occur at a lower dose, with increased exposure duration. Taken together, the key events and MOA are plausible and supported by the data. Other possible MOAs for liver effects from halauxifen

- methyl were also considered, butHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 23 of 126 the data did not support them. These included DNA reactivity, targeted gene expression of other genes such as CAR, PXR or PPARα, cytotoxicity/apoptosis, or other agents (infections, metals, statins, estrogens).
The MOA is considered relevant to humans based on the f act that the AhR pathway is conserved across species. PBPK modeling indicated that humans would have pre

- systemic exposure to halauxifen

- methyl, and halauxifen

- methyl was able to activate CYP1A1 genes in human hepatocytes. While there are in vitro data i ndicating that humans may be less sensitive than the rat to activation of AhR, a quantitative correlation to in vivo exposure is not possible. The data suggest, however, that humans are unlikely to show greater sensitivity than the rat.
4.4 Safety factor for Infants and Children (FQPA Safety Factor)
The halauxifen

- methyl risk assessment team has recommended that the FQPA Safety Factor be reduced to 1X . E xposure estimate s rely on data that reasonably and conservatively account for potential exposur es from dietary sources and occupational use. The dietary assessment is based on reliable data and will not underestimate exposure. There is no evidence of increased prenatal susceptibility to halauxifen

- methyl or pre

- /post

- natal susceptibility to XDE

- 729 acid. Residual concerns regarding lack of information on metabolism of halauxifen

- methyl during early post

- natal development are low because actual dietary exposure is expected to be very low and a conservative endpoint, protective of AhR activation i n the adult in the absence of actual liver toxicity, was selected for risk assessment.
4.4.1 Completeness o f the Toxicology Database The toxicology database for halauxifen

- methyl is considered adequate for assessment of developmental sensitivity/susceptibility and includes acceptable developmental toxicity (halauxifen

- methyl and XDE

- 729 acid), reproductive toxicity (XDE

- 729 acid), acute and subchronic neurotoxicity (XDE

- 729 acid) and immunotoxic ity (halauxifen

- methyl) studies. The bridging and mechanistic studies on halauxifen

- methyl, considered together with the reproductive toxicity study on XDE

- 729 acid, are considered adequate for assessment of post

natal effects.
4.4.2 Evidence of N eurotox icity There was no evidence of neurotoxicity in the available studies on halauxifen

- methyl and XDE

729 acid , including a cute and subchronic neurotoxicity studies conducted on XDE

- 729 acid

. In the acute neurotoxicity study, slightly reduced activity was o bserved but was considered an equivocal effect, based on lack of other observations, and was not considered evidence of a neurotoxic effect. The neurotoxicity data on XDE

- 729 acid are considered sufficient because systemic exposure is to the acid.
4.4.3 Evidence o f Sensitivity/Su sceptibility in the Developing o r Young AnimalHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 24 of 126 There was no evidence of increased prenatal quantitative or qualitative susceptibility. No developmental effects were observed in rats exposed to halauxifen

- methyl or rabbits expose d to XDE

- 729

- acid. In rats exposed to XDE

- 729 acid and rabbits exposed to halauxifen

- methyl, fetal toxicity (decreased fetal weight, delays in bone ossification) was observed only at doses that caused significant maternal toxicity (hepatotoxicity and mori bundity) . Toxicokinetic evaluations showed that maternal and fetal systemic exposure following exposure to halauxifen

- methyl was to XDE

- 729 acid, based on levels found in the blood. There was no evidence of increased postnatal quantitative or qualitative susceptibility to XDE

729 acid, based on lack of offspring or reproductive toxicity at doses causing parental toxicity in a rat two

- generation reproductive toxicity study. In the absence of reproductive toxicity data on halauxifen

- methyl, there is residu al uncertainty for increased postnatal susceptibility from dietary exposure to halauxifen

- methyl during early development (e.g. during weaning, via drinking water or solid foods) due to potential differences in liver metabolism and toxicity, which may resu lt from differential expression of liver Cyp1a1 expression (lower) or AhR levels (higher) in young offspring compared to adults. For example, decreased hepatic Cyp1a1 activity may result in a slower rate of metabolism, leading to greater hepatic and syste mic exposure to the parent compound. However, c oncern for increased susceptibility to halauxifen

- methyl is low for several reasons . The selected POD for all exposure assessments of halauxifen

- methyl is based on a dose that is well below doses that caus e minimal liver toxicity in adults. Halauxifen

methyl was demonstrated to be a relatively weak agonist of the AhR receptor, due to its rapid metabolism and excretion, and is unlikely to be a more potent inducer of Cyp1a1 expression in humans compared to ra ts. Furthermore, actual dietary exposure to halauxifen

- methyl is anticipated to be extremely low , due to low residue levels and degradation of parent compound in the environment.
4.4.4 Residual Uncertainty in the Exposure Datab ase Exposure to halauxifen

- methyl will not be underestimated in this assessment. There are no exposure data gaps. The dietary exposure assessment relies upon conservative assumptions and modelling data, together with a conservative dose and endpoint. The dietary risk estimates w ere based on tolerance level residues which reflect that measurable residues are not expected in any foods, no processing factors due to the lack of concentration in processed commodities , 100% crop treated assumption and modeled drinking water levels.
Re sidue chemistry data indicate that exposure in the diet (food and drinking water) is very low and primarily to XDE

- 729 acid, and that levels of halauxifen

- methyl itself are expected to be minimal due to environmental degradation

. Dietary exposure to resid ues from halauxifen

- methyl is primarily from drinking water. Residues in the proposed food crops show levels of halauxifen

- methyl and metabolites of c oncern that are below the LOQ.
4.5 Toxicity Endpoint and Point of D eparture Selections
4.5.1 Dose
- Respo nse AssessmentHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 25 of 126 Dietary exposure to halauxifen

- methyl and its metabolites/degradates is very low, primarily from drinking water contaminants. Residues in treated crops are below the LO Q . Separate dietary endpoints were selected for halauxifen

- methyl and XDE

- 729 acid due to their distinct toxicities. Most other, minor degradates are hydrolyzed at the methyl ester; their toxicity is expected to be similar to XDE

- 729 acid and they are considered together with the acid. Occupational endpoints were selected only for halauxifen

- methyl, the ai in end

- use products. No residential endpoints were selected because there are no proposed residential uses.
Halauxifen

- Methyl : For acute dietary exposure, no hazard was identified for halauxifen

- methyl acid that could be attributed to a single exposure. There are no single

- dose studies on halauxifen

- methyl, but liver toxicity from AhR activation, the most sensitive effect, is not considered a single dose effect.
No chronic studies are available for halauxifen

- methyl . For assessment of chronic dietary exposure, the POD of 3.0 mg/kg/day, based on increased liver Cyp1a1 expression (biomarker for AhR activation) at 10 mg/kg/day in the rat 90

- day oral toxicity study, was selected. This is an early event in the adverse o utcome pathway and therefore protective of liver toxicity for all exposure durations. The selected dose and endpoint are the most sensitive available and protective of all effects from halauxifen

- methyl, as well as from XDE

- 729 acid (see below). It is no ted that at the level of Cyp1a1 expression (52

- fold above controls) seen at 10 mg/kg/day, no actual adverse effects were identified, and that the mild liver effects identified at the study LOAEL of 53 mg/kg/day were associated with a significant increase ( >1500

- fold) in its expression. Selection of 3.0 mg/kg/day as the POD is protective of potential liver effects that may result from sustained long

- term exposure, since toxicity will not occur in the absence of the MIE. The selected POD is protective of de velopmental toxicity (NOAEL in rabbit 18.5 mg/kg/day, in rat, ≥323 mg/kg/day). An uncertainty factor (UF) of 100x was applied (interspecies factor of 10x and intraspecies factor of 10x). An additional UF for extrapolation from subchronic to chronic data (UF S ) was not added based on: (1) dose and endpoint were based on the MIE for AhR activation , a rapidly occurring event following exposure, and are therefore protective of liver toxicity from exposures of all durations; (2) rapid in vivo metabolism to XDE

- 729 acid, for which chronic data are available, and lack of bioaccumulation; (3) lack of progression of liver toxicity in rat 28

- and 90

- day studies and (4) low dietary exposure. The selected dose and endpoint are therefore protective of liver toxicity f rom exposures of all durations

. For short

- and intermediate

- term dermal (occupational) exposure, an endpoint of concern was not identified. A dermal toxicity study on halauxifen

- methyl was not conducted. No toxicity was observed in a rat 28

- day dermal t oxicity study on XDE

- 729 acid up to the limit dose; however, the dermal absorption of XDE

- 729 acid is not known. In an in vivo rat dermal absorption study on halauxifen

- methyl, toxicokinetic analysis showed that it was rapidly hydrolyzed to XDE

- 729 acid i n the blood following absorption; therefore, internal exposure is to the acid. Dermal absorption of halauxifen

- methyl was determined to be 24%. It is reasonable , given the similarities , to assume similar dermal absorption for XDE

- 729 acid and halauxifen

methyl. The toxicokinetic data for dermal absorption of halauxifen

- methyl support theHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 26 of 126 conclusion of XDE

- 729 acid as the toxic moiety. As noted above, no adverse effects were observed in the dermal toxicity study with XDE

- 729 acid. Considering these find ings together, HED does not expect toxic effects from dermal exposure to halauxifen

- methyl.
For short

- and intermediate

- term (occupational) inhalation exposure, an oral study was selected because inhalation data were not available. The POD of 3 mg/kg/d ay for increased liver Cyp1a1 expression at 10 mg/kg/day in the rat 90

- day toxicity study on halauxifen

- methyl, was selected. The study is of an appropriate duration. The selected dose and endpoint are protective of potential toxicity from ingestion of l arge, nonrespirable particles. Additionally, since the lung is a metabolically active tissue and susceptible to AhR activation and Cyp1a1 induction, the selected dose and endpoint are considered protective of potential portal

- of

- entry effects. Toxicity v ia the inhala tion route is assumed to be equivalent to toxicity via the oral route

. For inhalation risk assessment, the level of concern (LOC) is a MOE of 100, based on combined interspecies (10x) and intraspecies (10x) UFs. An additional UF DB for lack o f inhalation data was not added because the data were waived , halauxifen

- methyl has low volatility, and inhalation MOEs calculated using the oral POD were adequately protective (≥1000)

. XDE

- 729 Acid : For acute dietary exposure, no endpoint was identified for XDE

- 729 acid that could be attributed to a single dose. In the rat acute neurotoxicity study on XDE

- 729 acid, transiently decreased body weight was observed only at the limit dose of 2000 mg/kg. Slight decreases in activity level at 750 and 2000 mg/ kg did not show a dose

- response and were not considered to be toxicologically significant effects in the absence of other, associated neurological findings. For chronic dietary exposure to XDE

- 729 acid, the NOAEL of 20.3 mg/kg/day from the rat chronic ora l toxicity/carcinogenicity study was selected, based on kidney toxicity in females at the LOAEL of 101 mg/kg/day. This study is of the appropriate duration and is protective of all effects seen in other studies, including developmental effects observed in the rat . A NOAEL of 16.7 mg/kg/day was observed in the chronic oral dog study, based on kidney toxicity in females at 89.9 mg/kg/day. However, the NOAELs and LOAELs from the two studies are similar and the difference is considered to be the result of do se spacing, rather than increased sensitivity in the dog. An UF of 100x was applied (interspecies factor of 10x and intraspecies factor of 10x) for chronic dietary assessment of XDE

- 729 acid. The 10x FQPA SF was reduced to 1x, based on the completeness of the database and lack of pre

- and/or post

- natal susceptibility.
4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment When there are potential occupational and residential exposures to a pesticide, the risk assessment must address exposures from three major sources: oral, dermal and inhalation. It must be determined whether or not the individual exposures have the same toxicological effects. If they do, then they can be combined. For halauxifen

- methyl, there are no residential uses which could result in oral exposure , and a dose and an endpoint w as not identified for dermalHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 27 of 126 exposure. Therefore, only inhalation exposure from agricultural uses of halauxifen

- methyl will be quantitatively assessed , and no combined exposures are warranted

. 4.5.3 Cancer Classification and Risk Assessment Recommendation Separate cancer classifications were assigned to halauxifen

- me thyl and the metabolite XDE

- 729 acid, since each compound shows significantly different toxicity (with a MOA described for liver toxicity from halauxifen

- methyl).
XDE

- 729 acid is classified as “not likely to be carcinogenic to humans.” There was no evide nce of carcinogenicity in acceptable mouse carcinogenicity and rat chronic toxicity/carcinogenicity studies conducted on XDE

- 729 acid. Genotoxicity studies for XDE

- 729 acid were negative.
Halauxifen

- methyl is classified as “not likely to be carcinogenic to humans” at doses below those that induce liver Cyp1a1 expression

– a key early event in the MOA for liver toxicity and, with sustained activation of AhR from long

- term exposure, carcinogenicity . Genotoxicity studies for halauxifen

- methyl were negative. Rat chronic toxicity/carcinogenicity and mouse carcinogenicity studies were not conducted on halauxifen

- methyl itself, but when considered together, the rat subchronic dietary and mechanistic studies on halauxifen

- methyl, along with the chronic studies o n XDE

- 729 acid, provide sufficient information to assess potential carcinogenicity (see Sections 4.3.1 and 4.3.2). The POD for increased liver Cyp1a1 expression is protective of effects to the liver that, with time, may induce increased hepatocellular pro liferation and tumors . E xposure to other tissues will be to XDE

- 729 acid, which shows no evidence of carcinogenicity in the long

- term dietary studies.
4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment

Table 4 .5. 4. 1
. Summary of Toxicological Doses and Endpoints for Halauxifen
- Methyl for Use in Dietary and Non
- Occupational Human Health Risk Assessments
. Exposure/ Scenario Point of

Departure Uncertainty/FQP

A Safety Factors RfD, PAD,

Level of Concern for Risk Assessment Study and Toxicological Effects

Acute Dietary ( All P opulation s , including Infants and Children and Females age 13

- 49 ) No hazard from a single exposure was identified in the available database; therefore, no risk is

expected from this exposure s cenario.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 28 of 126 Table 4 .5. 4. 1
. Summary of Toxicological Doses and Endpoints for Halauxifen
- Methyl for Use in Dietary and Non
- Occupational Human Health Risk Assessments
. Exposure/ Scenario Point of

Departure Uncertainty/FQP

A Safety Factors RfD, PAD,

Level of Concern for Risk Assessment Study and Toxicological Effects

Chronic Dietary (All Populations) POD = 3.0

mg/kg/day UF A = 10 x

UF H = 10 x FQPA SF= 1 x Chronic RfD

= 0.03 mg/kg/day
cPAD = 0.03 mg/kg/day 90

- day oral toxicity in the rat

(halauxifen

- methyl) (MRID 48557 830) At the study NOAEL of 10 mg/kg/day, i ncreased hepatic Cyp1a1 expression (MIE for liver toxicity from AhR activation) was observed (endpoint selected for risk assessment)

. The lowest dose of 3.0 mg/kg/day was selected to be protective of potential long

- term effects from low

- level but sustaine d increased AhR expression in the liver. 1
S tudy L OAEL = 53 mg/kg/day, based on mild liver enlargement and pathology. Incidental Oral Short

Term ( all durations ) There are no residential uses for halauxifen

- methyl and therefore an endpoint was not

identified.
Dermal Short

Term ( all durations) There are no residential uses for halauxifen

- methyl and therefore an endpoint was not

identified. Inhalation ( all duration s) There are no residential uses for halauxifen

- methyl and therefore an endpoint was not

identified. Cancer (oral, dermal, inhalation) Classification: Not likely to be carcinogenic to humans at dose levels that do not induce

Cyp1a1 expression. The cRfD is considered protective of potential cancer effects because it protects for the MIE for hepatocellular proliferation ( AhR activation ) that, over time, may result in promotion of liver tumors

. Point of Departure (POD) = A data point or an estimated point that is derived from observed dose

- response data and used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL = no 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). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MIE = molecular initiating event 1 The POD selected for risk assessment was based on a non

- adverse finding, increased liver Cyp1a1 expression i n a rat 90

- day dietary study, which was observed below the study NOAEL of 10 mg/kg/day for liver toxicity. This effect is a biomarker for activation of AhR, which causes liver toxicity and hepatocellular proliferation. The POD reflected a mild increase in Cyp1a1 expression and was selected to be protective of potential liver effects resulting from chronic dietary exposure to halauxifen

- methyl. Other tissues and organs will not be exposed to halauxifen

methyl due to rapid conversion to XDE

- 729 acid. The P OD is protective of effects from exposure to XDE

- 729 acid.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 29 of 126 Table 4.5. 4 .2
. Summary of Toxicological Doses and Endpoints for Halauxifen
- Methyl for Use in Occupational Human Health Risk Assessments
. Exposure/ Scenario Point of

Departure Uncertainty

Factors Level of Concern

for Risk Assessment Study and Toxicological Effects
Dermal Short

Term (1

- 30 days) and Intermediate

Term (1

- 6 months) No hazard from dermal exposure was identified in the available database; therefore, no risk is

expected from this exposure scenario.

Inhalation Short

- Term (1

- 30 days) and Intermediate

term (1

- 6 months) POD = 3.0

mg/kg/day [ Absorption via the inhalation route assumed equivalent to oral absorption ] UF A =10x

UF H =10x Occupational

LOC for MOE = 100 90

- day oral toxicity in the rat

(halauxifen

- methyl) (MRID 48557830) At the study NOAEL of 10 mg/kg/day, i ncreased hepatic Cyp1a1 expression (MIE for toxicity from AhR activation) was observed the (endpoint selected for risk assessment). The lowest dose of 3.0 mg/kg/day was selected to be protective of potential long

- term effects from low

level but sustained increased AhR expression in the liver. 1
S t udy L OAEL = 53 mg/kg/day, based on liver enlargement and pathology at 53 mg/kg/day. Cancer (oral, dermal, inhalation) Classification: Not likely to be carcinogenic to humans at dose levels that do not induce

Cyp1a1 expression. The cRfD is considered protective of potential cancer effects because it protects for the MIE for hepatocellular proliferation ( AhR activation ) that, over time, may result in promotion of liver tumors

. 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 environmentall y relevant human exposures. NOAEL = no observed 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 member s of the human population (intraspecies). MOE = margin of exposure. LOC = level of concern. MIE = Molecular Initiating Event. 1 The POD selected for risk assessment was based on a non

- adverse finding, increased liver Cyp1a1 expression in a rat 90

- day di etary study, which was observed below the study NOAEL of 10 mg/kg/day for liver toxicity. This effect is a biomarker for activa tion of AhR, which causes toxicity and proliferation in the liver but also other tissues including the lung, which is metabolica lly active and contains Cyp1a1 . The POD reflected a mild increase in Cyp1a1 expression and was selected to be protective of potential lung effects resulting from inhalation exposure to halauxifen

- methyl. Other tissues and organs will not be exposed to hal auxifen

- methyl following inhalation exposure, due to rapid conversion to XDE

- 729 acid. The POD is protective of potential portal of entry effects in the respiratory tract from exposure to XDE

- 729 acid because it protects for the MIE

.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 30 of 126 Table 4 .5. 4.3
. Su mmary of Toxicological Doses and Endpoints for XDE
- 729 acid for Use in Dietary and Non
- Occupational Human Health Risk Assessments
. Exposure/ Scenario Point of

Departure Uncertainty/FQP

A Safety Factors RfD, PAD,

Level of Concern for Risk Assessment Study and Toxicological Effects

Acute Dietary ( All p opulation s , including Infants and Children and females age 13

- 49 ) No hazard from a single exposure was identified in the available database; therefore, no risk is

expected from this exposure scenario.

Chronic Dietary (All Populations) NOAEL =

20.3 mg/kg/day (females) UF A = 10 x

UF H = 10 x FQPA SF= 1 x Chronic RfD

= 0.20 mg/kg/day
cPAD = 0.20 mg/kg/day Rat two

- year dietary chronic

toxicity/carcinogenicity study NOAEL = 101/20.3 mg/kg/day [M/F] LOAEL = 404/101 mg/kg/day [M/F] based on increased mortality, altered urinalysis parameters, decreased body weight, increased kidney weights, adrenal zone glomerulosa hypertrophy, increased degeneration and regeneration of renal tubules and kidney stones, and bladder pathology in males; in females, hyperplasia of pelvic epithelium of the kidney. Incidental Oral Short

Term ( all durations ) There are no residential uses for XDE

- 729 acid and therefore an endpoint was not identified.

Dermal Short

Term ( all durations) There are no residential uses for XDE

- 729 acid and therefore an endpoint was not identified. Inhalation ( all duration s) There are no residential uses for XDE

- 729 acid and therefore an endpoint was not identified. Cancer (oral, dermal, inhalati on) Classification: Not likely to be carcinogenic to humans. Point of Departure (POD) = A data point or an estimated point that is derived from observed dose

- response data and used to mark the beginning of extrapolation to determine risk associated w ith lower environmentally relevant human exposures. NOAEL = no observed 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). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure.
5.0 Dietary Exposure and Risk Assessment
5.1 Metabolite/Degradate Residue Profile
5.1.1 Summary of Plant and Animal Metabolism StudiesHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 31 of 126 Appendix B summarizes the results of the plant and animal metabolism studies, including the structures of the metabolites and degradates. Studies depicting the met abolism of halauxifen

methyl in wheat and turnips as primary crops are adequate to delineate the residues of concern for the currently proposed uses. The studies were conducted at approximately the maximum application rate

. In the wheat metabolism study, residues of parent and all metabolites were found at levels below 10% of the total radioactive residue (TRR). Wheat m atrices harvested and analyzed for residues included immature forage and hay and mature straw and grain. I n the turnip metabolism study, residues above 10% of the TRR were found in turnip tops. Compounds that were found at this level were parent and the glucose conjugates of parent and XDE

- 729 acid. As the registrant is only proposing tolerances for residues in wheat and barley, the tolerance expression should include parent only at this time. In the confined rotational crop study, the TRR in all commodities at all plantback intervals was 0.001 ppm or less. Because of the very low TRR levels, none of the r adioactivity in the tissue samples was characterized further. Based on these very low residues , rotational crop tolerances do not need to be established. A field rotational crop study was not conducted and is not required for the uses associated with thi s petition. In the cattle feeding study, X11449757 was the only metabolite found above the LOQ in any livestock commodity analyzed (i.e., milk, muscle, liver, kidney, fat). Residues were found in liver, kidney, and fat. In the goat metabolism study, X 11449757 was found in significant levels along with its sulfate conjugate, XDE

- 729 acid, the sulfate conjugate of X11406790, and the N

or O

- glucuronic acid conjugate of X11406790. These metabolites were found in milk, liver, kidney, and fat. In the po ultry metabolism study, quantifiable levels of X11449757, XDE

- 729 acid, and the sulfate conjugate of X11406790 were found in liver and skin with fat. A poultry feeding study was not conducted and is not required for this use petition

. 5.1.2 Summary of Environmental Degradation In the environment, halauxifen

- methyl hydrolysis is pH dependent , with the compound being relatively stable to hydrolysis at neutral pH and the hydrolysis rate increasing at lower or higher pHs

. The estimated single first

- order (SFO) hydrolysis half

- lives for halaux ifen

- methyl at pH 4, 7, and 9 are approximately 81 days , 155 days, and 3 days, respectively. The only hydrolysis product reported is XDE

- 729 acid , which was produced quantitatively at each pH condition during the 30

- day study , suggesting that XDE

- 729 acid is stable to hydrolysis.
Halauxifen

- methyl degraded rapidly in an aqueous photolysis study, with a half

- life of less than 10 minutes, with major degradates of XDE

- 729 acid, as well as degradates 1, 2, 4, 10, 11 , and 14. In the soil photolysis study, r apid halauxifen

- methyl transformation was observed in the dark control (faster than the irradiated samples); therefore, the phototransformation of halauxi fen

methyl in soil could not be appropriately assessed as a route of dissipation in the soil u nder the conditions used in the submitted study. Halauxifen

- methyl is assumed to be stable to soil photolysis or at least photolysis is expected to be slower than aerobic soil metabolism. Transformation products observed in this soil photolysis study were XDE

- 729 acid andHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 32 of 126 X11449757. In an aerobic soil metabolism study, the major degradates observed were XDE

- 729 acid and X11449757, with half

- lives ranging between 1.1 to 5.3 days. In the anaerobic soil metabolism study, t he major degradates were XDE

- 729 acid and X11449757, with a half

- life of 3.1 to 7.1 days. The major degradates under anaerobic aquatic conditions included XDE

- 729 acid, X11449757, and X11406790.
Halauxifen

- methyl is characterized as moderately mobile; however, significant degradation of halauxifen

- methyl was observed during an adsorption and desorption equilibrium experiment. Therefore, the mobility of halauxifen

- methyl cannot be accurately assessed. Transformation products XDE

- 729 acid and X11449757 a re considered to be mobile to moderately mobile. Based on available data from a fish bioconcentration study, there is little concern of halauxifen

methyl bioaccumulation. All nine identified environmental transformation products have the potential to rea ch drinking water as they were major degradates in one or more biodegradation studies. Two degradates (XDE

- 729 acid and X11449757) were also observed in the terrestrial field dissipation study. Six degradates, degradates 1, 2, 4, 10, 11, and 14, were onl y detected in the aqueous photolysis study. It is unclear if the extraction procedures were suitable for quantitative recovery of these degradation products as well as minor transformation products. Deg 11 and 14 are different in structure than parent an d the other identified transformation products. These two transformation products no longer retain the picolinic acid moiety.
5.1.3 Comparison of Metabolic Pathways
Parent halauxifen

- methyl undergoes the same demethylation in wheat, turnips, goats, hens, and rats. In addition, many of the primary metabolites seen in the metabolism studies are also seen in the environmental degradation studies. Halauxifen

- methyl cont ains two methoxy groups (i.e.,

- OCH 3 groups). One of these methoxy groups forms the methyl ester moiety on the chemical and the other methoxy group is attached to the phenyl ring. The primary metabolic pathways involve conversion of either one or both o f these two methoxy groups to hydroxyl groups. When the methoxy group that forms the methyl ester is converted to a hydroxy group, the moiety becomes an acid. This compound is the XDE

- 729 acid metabolite. When the methoxy group on the phenyl ring is con verted to a hydroxyl group, the compound becomes a phenol (the X11406790 metabolite). Both of these metabolites can then be converted to the metabolite with both the acid and phenol moieties (the X11449757 metabolite). In plants and animals, these metabo lites can conjugate with various moieties, depending on the plant or animal species. Rat metabolism studies were performed with both the parent compound as well as the XDE

- 729 acid metabolite. Rats metabolized the compounds in a comparable manner. Absor ption and excretion were extensive and rapid. XDE

- 729 acid was the primary metabolite of the parent compound. When rats were dosed with XDE

- 729 acid, the limited metabolism that did occur was due to acyl

- glucuronidation or O

- demethylation to form X114497 57, followed by glucuronide or sulfate conjugation. Supplemental metabolism studies were done with dogs and mice. Metabolism was extensive and rapid in these species as well . Rats administered halauxifen

- methyl formed the same demethylated metabolites t hat were seen in the plant metabolism studies and in the environmental degradation studies: XDE

- 729 acid and X11449757.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 33 of 126 5.1.4 Residues of Concern Summary and Rationale Holman, E. 20 May 2014, D416125 HED’s Residues of Concern Knowledgebase Subcommittee (ROCKS) has recommended that the residue of concern in primary crops for both tolerance enforcement and risk assessment be parent halauxifen

- methyl only. The committee recommended that the residue of concern in ruminants and poultry for both tolerance enf orcement and risk assessment be the metabolite X11449757. These conclusions are summarized below in table 5.1.4.

Table 5.1.4
. Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression
. Matrix Residues I ncluded in Risk Assessment Residues I ncluded in Tolerance Expression for Compliance Monitoring Plants Primary Crop H alauxifen

- methyl H alauxifen

- methyl

Rotational Crop Not Applicable Not Applicable Livestock Ruminant X11449757 X11449757

Poultry X11449757 X11449757 Drinking Water Halauxifen

- methyl + XDE

- 729 Acid + X11449757 + X11406790 + Degradates 1, 2, 4, 10, 11, and 14 Not Applicable

Plants (Primary Crops): For dietary risk assessment and tolerance enforcement, the parent compound should be the only residue included as a residue of concern. Halauxifen

- methyl was the only quantifiable residue observed in the crop field trials, specifically in wheat straw and forage, with no quantifiable residues of halauxifen

- methyl in wheat grain or any barley matrix. XDE

- 729 acid was not quantifiable in any of the crop field trials. Rotational Crops: In the confined rotational crop study, the TRR in all commodities at a ll plantback intervals was 0.001 ppm or less. Because of the very low TRR levels, none of the RAC samples were characterized further. Therefore, the residues of concern in rotational crops are not applicable at this time, nor do rotational crop tolerance s need to be set at this time.
Livestock: For dietary risk assessment and tolerance enforcement, metabolite X11449757 (4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- hydroxyphenyl)

- pyridine

- 2

- carboxylic acid) should be the only residue of concern . X11449757 was the only quantifiable residue in the cattle feeding study, and was a major residue in the goat and poultry metabolism studies. Although quantifiable, the residues of XDE

- 729 acid, X11406790, and X11406790 conjugates were generally low (<0.01 ppm). Gi ven the anticipated livestock dietary burden associated with the proposed uses, X11449757 is c onsidered to be adequate for enforcement and risk assessment purposes. Should the dietary burden increase in the future due to proposed new uses, this decision m ay need to be revisited and additional data may be required, including a poultry feeding study. If poultry feeding studies are conducted in the future, then it is recommended that metabolites observed in the poultry metabolism study be analyzed, including XDE

- 729 acid, X11406790, and X11449757.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 34 of 126 Water: The ROCKS recommend ed including parent halauxifen

- methyl, as well as transformation products XDE

- 729 acid, X11449757, X11406790, degradates 1, 2, 4, 10, 11, and 14. P arent halauxifen

- methyl in the environm ent is rapidly metabolized primarily to XDE

- 729 acid, especially at low, environmentally relevant concentrations. Therefore , actual exposure is essentially to XDE

- 729 acid regardless of environmental exposure to either halauxifen

- methyl or the XDE

- 729 aci d. Therefore, grouping parent halauxifen

- methyl with XDE 729

- acid for drinking water is appropriate. From an exposure perspective, degradates 1, 2, 4, 10, 11, and 14 were only observed in the aqueous photolysis study. Since aquatic photolysis is only likely to occur in clear shallow surface water and on foliar surfaces, exposure to these transformation products is expected to be primarily in surface water rather than groundwater.
5.2 Food Residue Profile Dotson, D

. , 11 July 2014 , D4 16127
The submitted residue chemistry studies were generally well conducted and are adequate for supporting regulatory conclusions, establishing appropriate tolerance levels for enforcement, and for risk assessment

. The number of trials is sufficient and the geogra phic distribution of the trials captures the major wheat and barley growing regions in the U.S., per OCSPP Series 860 Guidelines

. Data from the trials are representative of worst

- case residues expected to result from application of halauxifen

- methyl accor ding to the proposed use patterns, and the residue data are supported by adequate analytical method performance and storage stability data

. For regulatory enforcement, a nalysis of residues can be accomplished through standard analytical techniques, and re sidues do not show any trends for dissipation during frozen storage. In the wheat field trials, parent halauxifen

- methyl was the only residue present in forage, grain, and straw (Table 5.2) . Residues of parent were below the LOQ of 0.01 ppm in all sample s of grain. Residues of XDE

- 729 acid were below the LOQ of 0.01 ppm in all samples of wheat forage, hay, grain, and straw. In the barley field trials, residues of both parent and XDE

- 729 acid were below the LOQ of 0.01 ppm in all samples of wheat forage, hay, grain, and straw. Under the currently proposed use pattern, tolerances are not needed for rotational crop commodities; however, HED notes that if future uses have higher application rates, limited field rotational crop data may be required

. The cattle feeding stud y is adequate and can be used to determine the need for tolerance s for ruminant commodities. No data deficiencies are associated with the cattle feeding stud y
. Based on the dietary burdens that were constructed and the results of the cattle feeding stud y and the hen metabolism study , tolerances are not needed for l ivestock commodities. There is no reasonable expectation of finite residues in livestock commodities (40CFR §180.6(a)(3). As noted previously, should the dietary burden inc rease in the future due to proposed new uses, this decision may need to be revisited and additional data may be required, including a poultry feeding study. Tolerances are not needed for wheat or barley processed commodities. In the wheat processing s tudy, an exaggerated application rate of 5x was used. All samples of unprocessed grain andHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 35 of 126 processed commodities contained residues below the limit of detection. In addition, in all wheat and barley field trials, residues were below the LOQ in all sample s of grain. Therefore, HED concludes that the processing data are adequate, and that tolerances are not needed for processed commodities.

Table 5.2 . Summary of Residues from the NAFTA Crop Field Trials with Halauxifen
- Methyl
. Proposed Use: 0.0088 lb a i /acre ( 9.8 g/ha) total application rate

1
- day PHI for Forage 21
- day PHI for Hay 60
- Day PHI for Grain and Straw

Crop Matrix Applic. Rate

lb ai/ A (g ai/ha) PHI

(days) Residues (ppm)

n Min Max LAFT HAFT 1 Median M ean . Std. Dev. 2
Wheat Wheat Forage 0.0087

- 0.0099 (9.7

- 11) 1 27 0.036 0.389 0.043 0.384 0.157 0.159 0.08 Wheat Hay 17

- 31 27 <0.01 0.026 <0.01 0.025 <0.01 0.011 0.004 Wheat Grain 42

- 73 27 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA Wheat Straw 42

- 73 27 <0.01 0.013 <0.01 0.012 <0.01 <0.01 0.0004 Barley Barley Hay 0.0089

- 0.0098 (9.98

- 11) 17

- 33 18 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA Barley Grain 43

- 83 18 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA Barley Straw 43

- 83 18 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA 1 : HAFT = Highest Average Field Trial. 2: Std. Dev.: Standard Deviation. Blank cell indicates that all field trial samples are below the LOQ. 5.3 Water Residue Profile Bohaty, R. 12 June 2014, D406773 EFED provided estimated drinking water concentrations (EDWCs) for the proposed uses (D406773, R. Bohaty, 06 / 12 /2014 ). EFED provided values for parent, acid metabolite, and the total toxic residues in both surface and groundwater. In all three cases (pare nt, acid metabolite, and total toxic residues) the groundwater values were higher than the surface water numbers. For parent halauxifen

- methyl , EFED used the PRZM

- GW Model and generated a maximum groundwater value of 0.007 ppb. For risk assessment, the d rinking water residues of concern include parent, the acid metabolite, and various other metabolites. For the XDE

- 729 acid dietary exposure assessment, all residues of concern were included as the total toxic residue , yielding an EDWC value of 19.5 ppb.

5.4 Dietary Risk Assessment Dotson, D

. 11 July 2014 , D417128Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 36 of 126 5.4.1 Description of Residue Data Used in Dietary Assessment As discussed previously, the toxicological effects caused by parent halauxifen

- methyl and the XDE

- 729 acid metabolite are different. Therefore, separate c hronic dietary exposure assessments were performed for the two compounds. For parent halauxifen

- methyl, an assessment was performed for food and drinking water. Due to the extremely low residues of halauxifen

- methyl in drinking water, a drinking water only assessment yielded no exposure (Doston D., D417128). The assessment that included food was based on tolerance level residues (LOQ

- level of 0.01 ppm) and the assumption that 100% of all barley, wheat, and triticale consumed will be treated with halauxifen

- methyl. For the acid metabolite, the assessment was based on drinking water alone because the acid metabolite is not a residue of concern in food, but it is a residue of concern in water. Processing factors were not used for any commodities in the assessments. The submitted processing study demonstrated that residues do not concentrate in cereal grain processed commodities. In addition, there are no defau lt processing factors for wheat or barley processed commodities.
5.4. 2 Dietary Exposure Risk Estimates Dietary exposure (food + drinking water) and risk estimates were developed for the following population subgroups: the general U. S. population, all i nfants (<1 year old), children 1

- 2, children 3

- 5, children 6

- 12, youth 13

- 19, adults 20

- 49, females 13

- 49, and adults 50+ years old

. Generally, HED is concerned when dietary risk estimates exceed 100% of the population

adjusted dose (PAD) for either acute or chronic exposure scenarios

. For halauxifen

- methyl and XDE

- 729 acid , all dietary risk estimates were well below 100% of either PAD (Table 5.4.3)

. In both of the chronic dietary exposure assessments that were performed, the general U.S. population and all population subgroups used < 1 % of the chronic population adjusted dose (cPAD).
Halauxifen

- methyl is classified as “not likely to be carcinogenic to humans at dose levels that do not induce liver toxicity or Cyp1a1 expression.” As a result, cancer ri sk is no t of concern for this chemical.
5.4. 3 Dietary Risk Assessment Summary Table

Table 5
. 4.3. Summary of Dietary Exposure and Risk Estimates for Parent Halauxifen
- Methyl and XDE
- 729 Acid. Population Subgroup Parent Halauxifen

- Methyl: Food Plus Water XDE

- 729 Acid Metabolite: Drinking Water Only Exposure Estimate, mg/kg/day % cPAD cPAD mg/kg/day Exposure Estimate

mg/kg/day % cPAD U.S. Population 0.000017 <1 0.20 0.000408 <1 All infants* 0.001010 <1 0.20 0.001053 <1 Children 1

- 2 yrs 0.000040 <1 0.20 0.000589 <1Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 37 of 126 Table 5
. 4.3. Summary of Dietary Exposure and Risk Estimates for Parent Halauxifen
- Methyl and XDE
- 729 Acid. Population Subgroup Parent Halauxifen

- Methyl: Food Plus Water XDE

- 729 Acid Metabolite: Drinking Water Only Exposure Estimate, mg/kg/day % cPAD cPAD mg/kg/day Exposure Estimate

mg/kg/day % cPAD
Children 3

- 5 yrs 0.000041 <1 0.20 0.000496 <1 Children 6

- 12 yrs 0.000030 <1 0.20 0.000358 <1 Youth 13

- 19 yrs 0.000018 <1 0.20 0.000298 <1 Adults 20

- 49 yrs 0.000015 <1 0.20 0.000407 <1 Adults 50

- 99 yrs 0.000012 <1 0.20 0.000403 <1 Females 13

- 49 yrs 0.000013 <1 0.20 0.000406 <1 *Most highly exposed population subgroup

6.0 Residential (Non
- Occupational) Exposure/Risk Characterization
At this time there are no proposed residential uses of halauxifen

- methyl.
6.1 Residential Bystander Post
- A pplication Inhalation Exposure Volatilization of pesticides may be a source of post

- application inhalation exposure to individuals nearby pesticide applications. The agency sought expert advice and input on issues related to volatilizatio n of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009, and received the SAP’s final report on March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html). The agen cy has evaluated the SAP report and has developed a Volatilization Screening Tool and a subsequent Volatilization Screening Analysis (http://www.regulations.gov/#!docketDetail;D=EPA

- HQ

- OPP

- 2014

- 0219). During Registration Review, the agency will utilize th is analysis to determine if data (i.e., flux studies, route

- specific inhalation toxicological studies) or further analysis is required for halauxifen

- methyl

. 6.2 Spray Drift Spray drift is a potential source of exposure to those nearby pesticide applications. This is particularly the case with aerial application, but, to a lesser extent, spray drift can also be a potential source of exposure from the ground application method s (e.g., aerial and groundboom ) employed for halauxifen

- methyl . The agency has been working with the Spray Drift Task Force (a task force composed of various registrants which was developed as a result of a Data Call

- In issued by EPA), EPA Regional Office s and State Lead Agencies for pesticide regulation and other parties to develop the best spray drift management practices (see the agency’s Spray Drift we bsite for more information). The A gency has also developed a policy on how to appropriately consider spray drift as a potential source of exposure in risk assessments for pesticides. The potential for spray drift will be quantitatively evaluated for each pesticide during the Registration Review process which ensures that all uses for that pesticide will be considered concurrently.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 38 of 126 The approach is outlined in the revised (2012) Standard Operating Procedures f or Residential Risk Assessment (SOPs)

- Residential Exposure Assessment Standard Operating Procedures Addenda 1: Consideration of Spray Drift. This document outlines the quantification of indirect non
- occupational exposure to drift.
7.0 Aggregate Exposure/Risk Characterization In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and risks from three major sources: food, drinking water, and residential exposures

. In an aggregate assessment, exposures from relevant sources are added together and compared to quantitative estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated

. When aggr egating exposures and risks from various sources, HED considers both the route and duration of exposure. As previously noted, there are no residential uses for halauxifen

- methyl at this time; therefore, dietary exposure is the only pathway appropriate for assessment

. A ggregate risk estimates are equivalent to the dietary risk estimates discussed in Section 5 and are below HED’s level of concern. 8.0 Cumulative Exposure/Risk Characterization Unlike other pesticides for which EPA has followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to halauxifen

- methyl or XDE

- 729 acid and any other substances, and halauxifen

- methyl or XDE

729 acid does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has not assumed that halauxifen

- methyl or XDE

- 729 acid has a common mechanism of toxicity with oth er 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 conc erning common mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/.
9.0 Occupational Exposure/Risk Characterization Figueroa, Z. 22 Ju ly 2014 , D4 16129 There is a potential for occupational exposure associated with handler activities (i.e., mixing, loading, and applying). Post

- application exposure is expected for workers re

- entering treated areas. Based on the proposed labels’ use pattern/directions (e. g., 1

- 2 applications per season), short

- term (1 to 30 days) and intermediate

- term (1 to 6 months) durations of exposure are expected for handlers. Long

- term exposures (greater than 6 months) are not anticipated.
9.1 Short
- and Intermediate
- Term Handler Risk It is HED policy to use the best available data to assess handler exposure

. Sources of generic handler data, used as surrogate data in the absence of chemical

- specific data, include the Pesticide Handlers Exposure Database Version 1.1 (PHED 1.1 ) and the Agricultural HandlerHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 39 of 126 Exposure Task Force (AHETF) database, or other registrant

- submitted occupational exposure studies

. Some of these data are proprietary (e.g., AHETF data), and subject to the data protection provisions of FIFRA

. Default assumption s established by HED ’s Science Advisory Council for Exposure were used for parameters such as body weight and area treated

. The maximum single application rate on the proposed label s was used to assess the handler exposure scenarios

. Refer to Table 3.3 for a complete use pattern description

. The inputs for area treated were based on information in ExpoSAC Policy 9.1.
Since no short

- or intermediate

- term dermal endpoints of concern were identified , a dermal exposure assessment was not conducted. Occupational assessments for halauxifen

- methyl are based on inhalation exposures only

. Table 9 .1 presents the estimated short

- and intermediate

- term inhalation risk estimates for occupational handlers. All occupational handler scenarios for the proposed uses resulted in inhalation risk estimates that are greater than the LOC of 1 00 (i.e., not of concern) at some level of PPE and/ or risk mitigation (i.e., baseline attire with no respirator; engineering control for aerial applications (enclosed cockpits)). The lowest inhalation MOE , identified for handlers conducting mixer/loader activities with a water dispersible granule ( WDG ) formulation for aerial applications , was 2,500 ; thus providing an ample margin to account for any uncertainties in t he route

- to

- ro ute extrapolation

. No additional PPE recommendations are needed on the proposed label s
.

Table 9 .1. Short
- and Intermediate
- Term Occupational Handler Exposure and Risk Estimates for Halauxifen
- methyl. Exposure Scenario Mitigation Level a Inhalation Unit

Exposure b (μg/lb ai) Application Rate c Area Treated d Inhalation Dose e (mg/kg/day) Inhalation

MOE f

Mixer/Loader Mixing/Loading WDG for Aerial Baseline 8.96 0.0091 lb ai/A 1 , 200 A 0.001220 2,500 0.0049 lb ai/A 0.000659 4,600 Mixing/Loading WDG for Groundboom Baseline 8.96 0.0091 lb ai/A 200 A 0.000204 15,000
0.0049 lb ai/A 0.000110 27,000 Mixing/Loading Liquids for Aerial Baseline 0.219 0.0058 lb ai/A 1 , 200 A 0.000019 160,000
Mixing/Loading Liquids for Groundboom Baseline 0.219 0.0058 lb ai/A 200 A 0.00000318 940,000
Applicator Applying Sprays for Aerial Application Engineering control (enclosed cockpits) 0.0049 0.0091 lb ai/A 1 , 200 A 0.00000067 4,500,000 0.0049 lb ai/A 0.00000036 8,300,000 0.0058 lb ai/A 0.00000043 7,000,000 Applying Sprays for Groundboom Application Baseline 0.34 0.0091 lb ai/A 200 A 0.00000774 390,000 0.0049 lb ai/A 0.00000416 720,000Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 40 of 126 Table 9 .1. Short
- and Intermediate
- Term Occupational Handler Exposure and Risk Estimates for Halauxifen
- methyl. Exposure Scenario Mitigation Level a Inhalation Unit

Exposure b (μg/lb ai) Application Rate c Area Treated d Inhalation Dose e (mg/kg/day) Inhalation

MOE f

0.0058 lb ai/A 0.00000493 610,000 Flagger Applying Sprays for Aerial Application Baseline 0.35 0.0091 lb ai/A 350 A 0.0000139 220,000 0.0049 lb ai/A 0.0000075 400,000 0.0058 lb ai/A 0.00000889 340,000 a. Mitigation level or PPE. b. Unit Exposures based on AHETF/PHED

. c. Application r ates are based on proposed use on label s ; for GF

- 2685 = 0.0091 lb ai/A, for GF

- 2686 and GF

- 2687 = 0.0049 lb ai/A; and GF

2688=0.0058 lb ai/A. d. Exposure Science Advisory Council Policy No. 9.1. e. Inhalation Dose (mg/kg/day) = Daily Unit Exposure (μg/ lb ai) x Application Rate (lb ai/acre) x A r ea Treated / Body Weight ( 80 kg). f . Inhalation MOE = HED ( 3.0 mg/kg/day ) / Inhalation Daily Dose (mg/ kg/ day). LOC = 1 00. 9.2 Short
- and Intermediate
- Term Post
- Application Risk
Inhalation There are multiple potential sources of post

- application inhalation exposure to individuals performing post

- application activities in previously treated fields. These potential sources include volatilization of pesticides and resuspension of dusts and/or particulates that contain pesticides. The A gency sought expert advice and input on issues related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009, and received the SAP’s final report on March 2, 2010 (http ://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html). The A gency has evaluated the SAP report and has developed a Volatilization Screening Tool and a subsequent Volatilization Screening Analysis (http://www.regulations.gov/#!docketDetail;D=EPA

- HQ

OPP

- 2014

- 0219). During Registration Review, the A gency will utilize this analysis to determine if data (i.e., flux studies, route

- specific inhalation toxicological studies) or further analysis is required for halauxifen

- methyl. In addition, the Agency is continuing to evaluate the available post

- application inhalation exposure data generated by the Agricultural Reentry Task Force. Given these two efforts, the Agency will continue to identify the need for and, subsequently, the way to incorporate occupati onal post

- application inhalation exposure into the agency's risk assessments. Although a quantitative occupational post

- application inhalation exposure assessment was not performed, an inhalation exposure assessment was performed for occupational/commerci al handlers. Handler exposure resulting from application of pesticides outdoors is likely to result in higher exposure than post

- application exposure. Therefore, it is expected that these handler inhalation exposure estimates would be protective of most occupational post

- application inhalation exposure scenarios.
DermalHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 41 of 126 Quantitative occupational post

- application risk estimates for agricultural workers following treatments to agricultural crops were not assessed, since no dermal endpoint was identified

. The REI specified on the proposed label is based on the acute toxicity of halauxifen

- methyl. Halauxifen

- methyl is classified as Toxicity Category IV via the dermal route and for skin irritation , and Toxicity Category III for eye irritation potential. It is not a skin sensitizer. Under 40 CFR 156.208 (c) (2) (iii), ai’s classified as Acute III or IV for acute dermal, eye irritation and primary skin irritation are assigned a 12

- hour REI. Therefore, the [156 subpart K] Worker Protection Statement interi m REI of 12 hours is adequate to protect agricultural workers from post

- application exposures to halauxifen

- methyl.
10.0 References Hansen, L. 2 March 2016 , D406773. Halauxifen

- M ethyl: Summary of the Mechanistic Data and a Framework Analysis for the Proposed Mode o f Action for Liver Toxicity. Hansen, L. 2 March 2016. D 430415. Halauxifen

- Methyl: Toxicology Data Evaluation Reports for Studies Submitted to Support the Proposed Registration of New Technical Active Ingredient (Joint Chemical Review with Canada and Australia). D otson, D. 11 July 2014 , D4 16127

. Halauxifen

- Methyl. Petition for the Section 3 Registration and the Establishment of Tolerances on Wheat, Barley, and Triticale. Summary of Analytical Chemistry and Residue Data Dotson, D

. 1 1 July 2014 , D416128. Halauxifen

- Methyl Chronic Aggregate Dietary (Food and Drinking Water) Exposure and Risk Assessment for the Section 3 Registration Action on Barley, Wheat, and Triticale

. Figueroa, Z. 22 July 2014 , D4 16128

. Halauxifen

- methyl. Occupational and Residential Exposure and Risk Assessment to Support the Registration of the New Active Ingredient on Wheat, Barley, and Triticale. Holman, E. 2 0 May 2014 , D4 16125

. Halauxifen

- methyl. Report of the Residues of Concern Knowledgebase Subco mmittee (ROCKS). Bohaty, R. 12 June 2014 , D4 06773

. Halauxifen

- methyl: New Chemical Drinking Water Assessment Safe, S., Lee, S.

- O. and Jin, U

- H. (2013) Role of the Aryl Hydrocarbon Receptor in Carcinogenesis and Potential as a Drug Target. Toxicol. Sciences online doi:100.1093/toxsci/kft128, June 14, 2013 Cohen, S.M. (2010) Evaluation of Possible Carcinogenic Risk to Humans Based on Liver Tumors in Rodent Assays: The Two

- Year Bioassay is No Longer Necessary. Toxicol. Pathol. 38:487

- 501 Nebert, D.W., Dalton, T.P, Okey, A.B. and Gonzalez, F.J. (2004) Role of Aryl Hydrocarbon Receptor

- mediated Induction of the CYP1 Enzymes in Environmental Toxicity and Cancer. J. Biolog. Chem. 279 (23):23847

- 23850, June 4, 2004.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 42 of 126 A ppendix A
. Toxicology Profile and Executive Summaries
A.1 Toxicology Data Requirements The requirements (40 CFR 158.340) for food use for halauxifen

- methyl are shown below 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 1 yes 1 yes 1 yes 1 yes 1 yes 1 yes

yes yes yes yes yes 870.3100 Oral Subchronic (rodent)

................................ ............... 870.3150 Oral Subchronic (nonrodent)

................................ ......... 870.3200 28

- Day Dermal

................................ .............................. 870.3250 90

- Day Dermal

................................ .............................. 870.3465 90

- Day Inhalation

................................ .......................... yes 1 yes 2 yes 2
no 3
no 3 yes

yes yes

-

- 870.3700a Developmental Toxicity (rodent)

................................ .. 870.3700b Developmental Toxicity (nonrodent)

............................ 870.3800 Reproduction

................................ ................................ . y es 1 yes 1 yes 2 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 (rat)

................................ ............ yes 2,4 yes 2 yes 2,4 yes 2 yes 2 yes

yes yes yes yes 870.5100 Mutagenicity

— Gene Mutation

- bacterial

..................... 870.5300 Mutagenicity

— Gene Mutation

- mammalian

................ 870.5xxx Mutagenicity

— Structural Chromosomal Aberrations

... 870.5xxx Mutagenicity

— Other Genotoxic Effects

....................... yes 1 yes 1 yes 1 yes 1 yes

yes yes yes 870.6100a Acute Delayed Neurotoxicity (hen)

............................... 870.6100b 90

- Day Neurotoxicity (hen)

................................ .......... 870.6200a Acute Neurotoxicity Screening Battery (rat)

................. 870.6200b 90

- Day Neurotoxicity Screening Battery (rat)

.............. 870.6300 Develop. Neurotoxicity

................................ ................. no no yes 2 yes 2
no -

- yes yes

- 870.7485 General Metabolism

................................ ...................... 870.7600 Dermal Penetration

................................ ........................ 870.7800 Immunotoxicity

................................ ............................. y es 1 yes 5 yes 5 yes

yes yes Special Studies for Ocular Effects

Acute Oral (rat)

................................ ........................... Subchronic Oral (rat)

................................ .................. Six

- month Oral (dog)

................................ .................. no no no -

-

- 1 Studies were conducted on both halauxifen

- methyl and XDE

- 729 acid 2 Study was conducted on XDE

- 72 9 acid 3 Study waiver granted 4 Requirement satisfied by 870.4300, Chronic/Oncogenicity study in the rat 5 Study was conducted on halauxifen

- methylHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 43 of 126 A.2 Toxicity Profiles

Acute Toxicity of Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./Study Type MRID Results Toxicity Category 870.1100 Acute oral toxicity (rat) 48557814 LD 50 > 5000 mg/kg IV 870.1200 Acute dermal toxicity (rat) 48557816 LD 50 > 50 00 mg/kg IV 870.1300 Acute inhalation toxicity (rat) 48557818 Study not submitted based on inability to generate test atmosphere. -

870.2400 Acute eye irritation (rabbit) 48557820 Mild eye irritant III 870.2500 Acute dermal irritation (rabbit) 48557822 Not a dermal irritant IV 870.2600 Skin sensitization (guinea pig) 48557824 Not a dermal sensitizer NA N A = Not Applicable

Acute Toxicity of XDE
- 729, acid (metabolite of halauxifen
- methyl, or XDE
- 729 methyl) Guideline No./Study Type MRID Results Toxicity Category 870.1100 Acute oral toxicity (rat) 48557834 LD 50 > 5000 mg/kg IV 870.1200 Acute dermal toxicity (rat) 48557815 LD 50 > 5000 mg/kg IV 870.1300 Acute inhalation toxicity (rat) 48557817 Study not submitted based on inability to generate test atmosphere. -

870.2400 Acute eye irritation (rabbit) 48557819 Mild eye irritant III 870.2500 Acute dermal irritation (rabbit) 48557821 Not a dermal irritant IV 870.2600 Skin sensitization (guinea pig) 48557823 Not a dermal sensitizer NA NA = Not ApplicableHalauxifen

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Page 44 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

Subchronic Studies 870.3050 28

- day oral toxicity (mice) Study conducted only for

XDE

- 729 acid

870.3050 28

- day oral toxicity (F344/DuCrl rats) 48557826 (2011)

Acceptable/guideline Dietary concentrations equivalent to: 0, 10.8, 55.6, 296, or 822 mg/kg/day for males 0, 10.7, 55.5, 277, or 825 mg/kg/day for females NOAEL = 10.8/55.5 mg/kg/day (M/F) LOAEL = 55.6 mg/kg/day in males, based on increased liver weight, hepatocellular hypertrophy, increased mitotic figures in hepatocytes, hepatocellular vacuolization (consiste nt with fatty change) and hypertrophy of thyroid gland follicular cells. The female LOAEL is 277 mg/kg/day, based on decreased body weight gains and food consumption, increased liver weight, increased cholesterol levels and hepatocellular hypertrophy. At the highest dose slight thymic atrophy, acinar cell hypertrophy of th e submandibular salivary gland, hyperplasia of erythroi d cells in the bone marrow, increase d incidence of very slight extramedullary hematopoiesis (e rythroid cell) in the spleen, and decr eased size of the cervix, uterus and vagina (females) were observed. Toxicokinetics: XDE

- 729 acid was present in blood, but not halauxifen

- methyl. Less than 0.1% AD halauxifen

- methyl was present in urine; remainder excreted as XDE

- 729 acid. Saturation of either absorption or metabolism possible. 870.3050 28

- day oral toxicity (Beagle dog) Study conducted only for

XDE

- 729 acid formHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 45 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

870.3100 90

- Day oral toxicity (F344/DuCrl rats) 48557830 (49124405,

amended report) (2011) Acceptable/guideline Dietary concentrations equivalent to: 0, 3.1, 10.3, 53.4, 270 or 513 mg/kg/day in males and 0, 3.0, 10.1, 52.3, 263 or 504 mg/kg/day NOAEL = 10.0/10.1 mg/kg/day [M/F] LOAEL = 53.4/52.3 mg/kg/day [M/F], based on increased hepatocellula r vacuolization (males) and increased cholesterol and liver weights (females). At 270/263 mg/kg/day, lower % neutrophils and increased lymphocytes (females), increased cholesterol (males), increased absolute and relative liver weight, hypertrophy with cy toplasmic eosinophilia, increased absolute and relative thyroid weight and thyroid hypertrophy were observed. At 513/504 mg/kg/day, decreased body weight and weight gain and decreased thymic weight and atrophy (females) were observed. Toxicokinetic studies : Halauxifen

- methyl showed dose

- dependent levels in liver at all doses, and in blood ≥10 mg/kg/day. XDE

- 729 acid metabolite w as present as the major moiety in blood, liver, urine, along with other minor conjugated metabolites. Data suggest nonlinearity in metabolism/elimination of halauxifen

methyl, with increasing conjugation of metabolites as dose increased, and induction of hepatic metabolic enzymes. Gene expression studies: Cyp1a1 expression in liver showed a dose

- related increase. At 3 mg/kg/day e xpression was slightly higher but similar to controls (3.6 to 5.7

- fold) but showed a more pronounced increase (52

- 149

- fold) at the NOAEL of 10 mg/kg/day and a significant increase (1495

- 2268

- fold) at the LOAEL of 53 mg/kg/day. 870.3150 90

- Day oral toxicity (Beagle dog) Study conducted only for

XDE

- 729 acid form.

870.3200 2 8

- or 90

- d ay dermal toxicity (rat s ) Study conducted only for

XDE

- 729 acid form. Waiver for XDE

- 729 methyl granted based on lack of systemic exposure to methyl ester following dermal exposure (acid form found in blood in dermal absorption study). 870. 28

- or 90

- day inhalation toxicity (rats) Waiver granted due to

inability to generate test atmosphere.

Developmental and Reproduction StudiesHalauxifen

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Page 46 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

870.3700a Prenatal developmental probe study [(Crl:CD(SD) rats ] 4 8 55 7881 ( 2012 )

Acceptable/ non guideline Dietary concentrations of 0, 521, 2083, 4167 or 833 ppm, equivalent to 0, 40.4, 169, 303 or 490 mg/kg/day Administered GD 6 through 21 Maternal NOAEL = 169 mg/kg/day LOAEL = 303 mg/kg/day based on decreased food consumption and body weight gains. Dams given 490 mg/kg/day were sacrificed on GD 9 due to excessive body weight loss. Developmental NOAEL/LOAEL = not determined, fetal examination not performed. There were no signifi cant cesarean findings.
870.3700a Prenatal developmental study [Crl:CD(SD) rats ] 48557836 (2012)

Acceptable/guideline Dietary concentrations of 521, 2083 or 4167 ppm, equivalent to 0, 41.1, 159 or 323 mg/kg/day Administered GD 6 through 21 Materna l NOAEL = 159 mg/kg/day LOAEL = 3 2 3 mg/kg/day based on decreased body weight gains and food consumption, increased liver weight and hepatocyte histopathology (altered centrilobular and midzonal cytoplasmic homogeneity and hepatocellular vacuolization consist ent with fatty change)

. Developmenta l NOAEL ≥ 3 23 mg/kg/day No dose related effects were seen, thus the developmental LOAEL was not determined. 870.3700b Prenatal developmental probe study (NZW rabbit s ) 48557839 (2012)

Acceptable/nonguideline Dietary concentrations of 0, 521, 2083, 4167. 8333, 10417, 15625 or 24,500 ppm, equivalent to: 0, 21.5, 83.3, 157, 244, 371, 426 or 727 mg/kg/day Administered GD 7 through 28 Matern a l NOAEL = 157 mg/kg/day LOAEL = 244 mg/kg/day based on decreased body weight, wei ght gain and food consumption

. Dams given 727 mg/kg/day were prematurely sacrificed on prior to GD 15 due to excessive weight loss. Developmenta l NOAEL/LOAEL = not determined, fetal examination not performed. There were no significant cesarean findings. No dose related effects were seen, thus the developmental LOAEL was not determined.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 47 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

870.3700b Prenatal developmental (NZW rabbit s ) 48557840 ( 2012 )

Acceptable/guideline Dietary concentration of 0, 122, 391 or 1539 ppm, equivalent to 0, 5.8, 18.5 or 71.6 mg/kg/day Administered GD 7 through 28 Materna l NOAEL = 5.8 mg/kg/day LOAEL = 18.5 mg/kg/day based on increased liver weight and hepatocellular histopathology (hypertrophy with increased cytoplasmic eosinophilia of periportal hepatocytes, increase d number of mitotic figures in hepatocytes, increased altered cytoplasmic homogeneity of centrilobular/midzonal hepatocytes

. At 71.6 mg/kg/day, increased panlobular vacuolization of hepatocytes consistent with fatty change was also observed

. Developmenta l NOAEL = 18.5 mg/kg/day LOAEL = 71.6 mg/kg/day, based on decreased fetal body weights and delayed ossification of the pubis. 870.3800 Reproduction and fertility effects ( rats) Study conducted on XDE

729 acid

Chronic Toxicity Studies 870.4100b Chronic toxicity (Beagle dog s ) Study conducted on XDE

729 acid

Chronic/Carcinogenicity Studies 870.4200b Carcinogenic ity (mic e) Study conducted on XDE

729 acid

870.4300 Chronic/ Carcinogenicity

- ( rat s) Study conducted on XDE

729 acid

Mutagenicity Studies Gene Mutation 870.5100 Bacterial Reverse Mutation Test (pre

incubation assay) 48557846 (2011)

Acceptable/guideline 0, 50, 10, 500, 1 5 00, or 5000 μg/plate +/

- S9 Negative in Salmonella typhimurium strains TA 1535, TA1537, TA98, and TA100 and Esc h erichia coli Wp2 uvrA up to the limit concentration with evidence of compound insolubility but no cytotoxicity

.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 48 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

Gene Mutation 870.5100 Bacterial Reverse Mutation Test (plate

incorporation assay) 48557845 (2011)

Acceptable/guideline 0, 50, 10, 500, 1 5 00, or 5000 μg/plate +/

- S9 Negative in Salmonella typhimurium strains TA1537 , TA98, and TA100 and Esc h erichia coli Wp2 uvrA up to the limit concentration with no evidence of compound insolubility or cytotoxicity

.

Gen e Mutation 870.5300 In vitro mammalian cell forward gene mutation, Chinese hamster ovary (CHO) cells , HGPRT locus

48557851 (2012) Acceptable/guideline 0, 15.625, 31.25, 62.5, 125, 250 or 500 μg/m L

- S9 activatio n and 0, 18.75, 37.5, 75, 150, 300 or 500 μg/m L +S9 activation Negative for the induction of mutant colonies over background up to precipitating concentrations

Gene Mutation 870.5300 In vitro mammalian cell forward gene mutation, Chinese hamster ovary (CHO) cells , HGPRT locus

48557849 (2011) Acceptable/guideline 0, 3.13, 6.25, 12.5, 25 or 50 μg/mL+/

- S9 activation Negative for the induction of mutant colonies over background up to precipitating concentrations.

Cytogenetics 870.5375 In vitro mammalian cell chromosomal aberration assay , human peripheral lymphocytes 48557854 (2012)

Acceptable/guideline 0, 15.624, 31.25, 62.5, 125, 250 or 500 μg/mL +/

- S9 activation Negative for induction of chromosomal aberrations over background up to cytotoxic and precipitating concentrations.

Cytogenetics 870.5375 In vitro mammalian cell chromosomal aberration assa y , primary lymphocyte cultures (Sprague Dawley rats) 48557853 (2012)

Acceptable/guideline 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μg/mL

- S9 activation and 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μg/mL + S9 activation Negative for induction of chromosomal aberrations over background up to precipitating concentrations.

Neurotoxicity Studies
870.6200a Acute Neurotoxicity Screening Battery (rats) Conducted using XDE

- 729

acid

870.6200b Subchronic Neurotoxicity Study (rats) Conducted using XDE

- 729

acid
Other StudiesHalauxifen

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Page 49 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

870.7485 Metabolism and pharmacokinetics (rats) 48557860 (2010)

Acceptable/guideline See MRID 48557860, XDE

729 870.7600 Dermal penetration in vivo (rats) 4 8557911 ( 2011 )

Acceptable/guideline [ 14 C]

- XDE

- 729 methyl in EC Formulation GF

- 2573 was applied to shaved rat skin as concentrate, field 1 and field dilution 2 (75, 1.5 and 0.25 μg/cm 2 , respectively)

. Exposure time was 10 hours , followed by measurement of absorption at 24, 48, 72 or 144 hr (concentrate) and 24, 48, 96 and 192 hr (dilutions). Dermal absorption increased with dose but not proportionally. The percentage of the dose absorbed decreased with the increasing administered dose. The total body burden could be ex pected to rapidly decrease due to excretion via urine and feces. Mean dermal absorption for the 10

- hour exposure interval (at 144/192 hr post

- exposure ) was 13, 15 and 24 % in the concentrate, field dilution 1 and field dilution 2 dose groups, respectively

. Blood measurements demonstrated that systemic exposure was to XDE

- 729 acid due to rapid hydrolysis of the parent following absorption.

Nonguideline study Dermal penetration, comparative in vitro study on rat and human skin 48557910 (2011)

Acceptable/non guideline
[ 14 C]

- XDE

- 729 methyl in EC Formulation GF

- 2573 was applied to rat and human skin samples as concentrate, field 1 and field dilution 2 (75, 1.5 and 0.25 μg/cm 2 , respectively)

. Exposure time was 10 hours , followed by measurement of absorption at 24 hr. Percent dermal absorption for rat skin samples was 4.9 , 41 .1 and 4 2.7 % of the applied dose. Percent dermal absorption for human skin samples was 0. 8 , 1 4.9 and 26 .5 % of the applied dose. The ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 3.0 and 2. 2 (24 hr in vivo absorption used for comparison). The ratio of human to rat in vitro absorption (highest to lowest concentration tested) is 0.1 6 , 0.3 6 and 0.6 2 , respectively.

870.7800 Immunotoxicity ( F344/D uCrl rat , females only ) 4855786 4 (201 2 )

Acceptable/guideline Dietary concentrations of 0, 1 47 , 64 3 or 6323 ppm , formulated to provide 0, 10, 52 , or 500 mg/kg/day Immunotoxicity NOAEL = 500 mg/kg/da y Immunotoxicity LOAEL = No dose related effects were seen, thus the immunotoxicity LOAEL was not determined. Systemic NOAEL = 52 mg/kg/day Systemic LOAEL = 500 mg/kg/day based on decreased thymus weight and increased liver weight.

Non

- Guideline StudiesHalauxifen

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Page 50 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

Hepatic gene expression and biomarker analyses (male F344/DuCrl rats) 48557879 (2012)

Acceptable/nonguideline Dietary concentrations formulated to provide 0, 766 mg/kg/day XDE

- 729 methyl ester (halauxifen

methyl) or 672 mg/kg/day XDE

- 729 acid, administered for 7 da ys XDE

- 729 acid toxicity : There were no effects on the liver or thyroid. Decreased body weight and food consumption were observed. XDE

- 729 methyl toxicity : Observed effects included increased liver weight, very slight panlobular hypertrophy of hepato cytes, very slightly increased numbers of mitotic figures in hepatocytes, increased thyroid weight, very slight hypertrophy of thyroid gland follicular cells. Toxicokinetics and gene expression : Test material was present in liver and blood. Most of excr eted test material was as XDE

- 729 acid, with very little XDE

729 methyl. In liver and urine, low levels of an acyl glucuronide conjugate of XDE

- 729, O

- demethyl XDE

729, sulfate conjugate of O

- demethyl XDE

- 729 and glucuronide conjugates of O

- demethyl XDE

- 7 29 and XDE

- 729 methyl were identified. Thyroid effects appeared to be secondary to liver alterations, based on observed activation of the AhR signaling pathway genes by XDE

- methyl, based on increased hepatic Cyp1a1 levels. Animals treated with XDE

- 729 ac id did not show induction of liver metabolic genes and animals treated with XDE

- 729 methyl did not show significant activation of enzymes of the CAR pathway, or the PXR or PPAR

- α signaling pathways. Seven day dietary toxicity study probe study [Crl:CD(ICR) mice] 48557872 (2012)

Acceptable/nonguideline Dietary concentrations formulated to supply: 0, 250 or 778 mg/kg/day in males and 0, 276 or 863 mg/kg/day in females, Administered for seven days Males and females at 778/863 mg/kg/day showed inc reased liver weights but no histopathological changes and increased Cyp1a1 mRNA expression (AhR activation). Parent and XDE

- 729 acid generally exhibited linear kinetics in liver, blood (6 am) & urine. XDE

- 729

- methyl was detected at low levels in ~50% of blood samples at all doses but represented ≤1% of the XDE

729 levels (0.03

- 0.07 μg/mL). XDE

- 729 methyl was only in urine at the high dose (0.20

- 0.24 μg/mL). Acid was present in all treated animals at 100 and 9000

- fold higher than methyl for blood and urine , respectively. In liver, methyl was 50 to 100

- fold higher than blood (1.39

- 4.95 μg/g). In liver, acid was 2

- fold higher than the methyl. Levels of both XDE

- 729 methyl and XDE

- 729 acid were generally lower in female mice compared to males in blood (6 am), liver and urine; the only exceptions were the terminal blood concentrations, where slightly higher XDE

- 729 methyl and XDE

- 729 acid levels were seen in females. Kinetics were similar in male mice and rats. The rat is more sensitive than the mouse to XDE

- 7 29 methyl

induced hepatic responsesHalauxifen

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Page 51 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

28

- day dietary study on molecular and cellular changes in the liver [ male rats] 48557873 (2012)

Acceptable/nonguideline Dietary concentrations of 0, 57.7, 198, 1036 or 5156 ppm formulated to provide 0, 3, 10, 55 or 271 mg/kg/day Administered for 28 days plus additional recovery groups were given 0 or 10.3 mg/kg/day and a 4

- day recovery period, or 0, 53.7 or 277 mg/kg/day with a 28

- day recovery period. No treatment

- related findings were observed on survival, clini cal signs, body weights, food consumption or kidney weights. At ≥3 mg/kg bw/day: No quantifiable halauxifen

methyl in liver at 3 mg/kg bw/day, ≤0.36 μg/g at 10 and 52 mg/kg bw/day, 3.0 μg/g at 261 mg/kg bw/day At ≥55 mg/kg bw/day: increased liver weight, increased Cyp1a1 mRNA expression (684, 121800

- fold), increased hepatocellular hypertrophy with increased cytoplasmic eosinophilia, hepatocellular vacuolation, mitotic figures. At 271 mg/kg bw/day: Marginal ↑ Brd U uptake in liver, ↑ Cyp1a2 (22

- fold), Ugt1a6 (11

- fold) & Cyp2b1 (8

- fold) mRNA expression Recovery: Complete reversal of changes in liver weight, liver histopathology, Cyp gene expression & quantifiable liver concentrations of methyl & acid TK: Low levels of methyl in liver, which became supralinear between 54.8

- 271 mg/kg/day. Levels of methyl <LLQ in all blood samples; acid was present in all blood samples (minor metabolites not measured). Blood levels of methyl were sub

- linear at 271 mg/kg/day. In urine, ~0.05% dose was excreted as methyl and ~50% dose was excreted as acid. Total amount of acid in urine was sub

- linear at 271 mg/kg/day. Comparative in vitro AhR transactivation (Luciferase reporter and competition ligand binding assays) [ Mouse (Hepa 1.1), human (Hep G2 40/6) liver cell lines ] 48557878 (2012)

Acceptable/nonguideline 1) Cell

- based Luciferase reporter assay: 0.001, 0.01, 0.1, 1, 10, 50, 100 μM (XDE

- 729 methyl, ITE); 0.01, 0.1, 1, 10, 50 μM (XDE

- 729 methyl ultrapure) 2) Competition lig and

binding assay: 0.01, 0.1, 1, 10, 50 μM (XDE

- 729 methyl ultrapure) In cells exposed to XDE

- 729 methyl at ≥10 μM , a weak increase in AhR transactivation activity was observed in mouse Hepa 1.1 reporter cell line only, compared to positive control ITE (not due to an impurity)

. XDE

- 729 methyl ultrapure (99.7%) is an AhR ligand that weakly competes with the photoaffinity ligand for the rat Ah receptor (EC 50 = 1.5x10

- 5 (XDE

- 729 methyl), 8.4x10

- 7 (ITE)). No AhR transactivation was observed with XDE

- 729 acid

.Halauxifen

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Page 52 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

In vitro AhR nuclear receptor activation & Cyp1a mRNA expression [ Mouse, Rat, Human hepatocytes ] 48557876 (2012)

Acceptable/nonguideline 0.01, 0.1, 1.0, 10, 30, 100 μM XDE

- 729 methyl for 0, 2, 4, 8, 12, 24 h Positive controls: 3

- methylcholanthrene (3

- MC; 0

- 5 μM); phenobarbital (PB; 1 μM) Exposure to XDE

- 729 methyl at concentrations ≥30 μM caused l imited induction of Cyp1a1 and Cyp1a2 in mouse and human hepatocytes at multiple time

- points up to 24 h compared to 3

- MC. Human hepatocytes were less resp onsive than mouse and rat hepatocytes to Cyp1a1 induction but more responsive to Cyp1a2 induction compared to mice. Peak expression occurred from 8

- 12 h. Sensitivity to Cyp1a induction: Rat>Mouse>Human ( Cyp1a1 > Cyp1a2 ) 3

- MC: Dose responsive ↑ Cyp1a1 and Cyp1 a2 in all species. Cyp1a1 was more responsive compared to Cyp1a2 with the mouse and human, but Cyp1a2 was induced to a greater extent in the rat. For Cyp1a1 induction, mouse was the most sensitive, followed by the human where all but 1 donor had a higher C YP1A1 induction than rat. Rat was most responsive to 3

- MC with respect to Cyp1a2 induction, human was the next most responsive and mouse had the least amount of Cyp1a2 induction. PB: Induction of Cyp2b10 (mouse; modest response), Cyp2b6 (rat) and CYP2B6 (human) in hepatocytesHalauxifen

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Page 53 of 126 Subchronic, Chronic, and Other Toxicity Studies for Halauxifen
- methyl (XDE
- 729 methyl) Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

Comparative in vitro methyl ester hydrolysis, PBPK modeling [Mouse, rat and human tissues; whole blood and synthetic gastric fluid (SGF)] 48557874 (2012)

Acceptable/nonguideline
Liver S9: 0.5, 1.0, 2.0, 5.0, and 10.0 μM XDE

- 729 methyl

- sampling at 0, 3, 8, 15, and 30 minutes, post

- fortification (rat, human) 0, 3, 8, 15, and 30, 60, and 120; 0.25, 0.5, 1.0, 5.0, and 10.0 μM

- 0, 3, 8, 15, 30, 60, and 120 minutes (mouse)
Whole blood: 1 and 100 μM XDE

- 729 methyl

- 0, 0.08 , 0.25, 0.5, 1, 2, 4, 8, 24 hours, post

f ortification. 0.25, 0.5, 1, 5, and 10 μM

- 0, 0.05, 0.13, 0.25, 0.5, 1, and 2 hours post

- fortification SGF: 1, 100 μM XDE

- 729 methyl at pHs 1.2, 3.0, 4.0

- 0, 0.08, 0.25, 0.5, 1, 2, 4, 8 and 24 hours, post

- fortific ation Rapid hydrolysis to XDE

- 729 acid by liver S9 was observed in all species, with the relative rate of metabolism in order of (fastest to slowest) human>> rat>mouse. In human liver S9, XDE

- 729 methyl was not present at >LOQ after 8 min of incubation. X DE

729 methyl was metabolized in whole blood by 1st order kinetics (fastest to slowest): rat>mouse>>human The rate of metabolism in synthetic gastric fluid was the slowest. The fastest rate of conversion at pH 1.2 (i.e. pH of human GIT) = t1/2 100 h was mu ch slower than liver S9 & whole blood. The predicted hydrolysis of XDE

- 729 methyl was most dependent on the rate of metabolism in liver tissue for both species (~60% of total hydrolysis in rat and >95% in human). Model simulations (adapted to simulate a 2 8

- day exposure) indicate that XDE

- 729 methyl and XDE

- 729 acid achieve steady

- state concentrations soon after exposure is begun, the levels clear rapidly from blood and liver following the cessation of exposure, and that neither XDE

- 729 methyl nor XDE

- 729 w ould be expected to accumulate in either liver or blood following exposures to XDE

- 729 methyl. Model

derived peak concentrations (and 24 h AUCs) of XDE

729 methyl in liver and blood are comparable between rats and human. Peak concentrations from simulat ed 28 d exposure to XDE

- 729 methyl at 0.001 mg/kg bw/day (relevant exposure level based on residue data) resulted in levels 10000

- 15000

- times lower than LOQ (LOQs: in blood ~0.01 μg XDE

- 729 methyl/g; in liver ~0.04 μg XDE

729 methyl/g).Halauxifen

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Classification /Doses Results

Subchronic Studies 870.3050 28

- day oral toxicity [ Crl: CD

- 1(ICR) mice] 48557827 (2009)

Acceptable/guideline Dietary concentrations of 10, 50, 250 or 1000 ppm, equivalent to: 0, 10.2, 54.0, 269 or 1025 mg/kg/day for males and 0, 9.9, 48.7, 241 or 958 mg/kg/day for females NOAEL = 269/241 mg/kg/day [M/F] LOAEL = 1025/958 mg/kg/day [M/F], based on decreased numbers and mean volume of red blood cells, decreased alanine aminotransferase activity, decreased weight and an increased incidence of tubule degeneration in the kidney, and increased incidences of m ultifocal karyomegaly and focal necrosis in the liver.

870.3050 28

- day oral toxicity (F344/DuCrl rats) 4855782 5 (2009)

Acceptable/guideline Dietary concentrations equivalent to: 0, 10.5, 52.7, 270 or 734 mg/kg/day for males 0, 10, 50.3, 250 or 982 mg/kg/day for females, respectively NOAEL = 270/250 mg/kg/day M/F LOAEL = 734/982 mg/kg/day M/F , based on decreased body weight, body weight gains and food consumption, increased urinary volume, decreased urine specific gravity, cloudy u rine and phosphate crystals in urine sediment (females only), increased relative kidney (males only) weights, renal histopathology (degeneration and hypertrophy of renal tubules and vacuolization of collecting duct epithelium) and splenic extramedullary he matopoiesis. 870.3050 28

- day oral toxicity (Beagle dog) 48557871 (2010)

Acceptable/guideline Dietary concentrations of 0, 300, 3000, 15,000 (females only) or 30,000 ppm (males only), equivalent to: 0, 11, 80 or 635 mg/kg/day in males 0, 9, 85 or 323 mg/kg/day in females NOAEL = 11/85 mg/kg/day [M/F] LOAEL = 80/323 mg/kg/day [M/F], based on degeneration and regeneration of renal tubules, and in females, body weight loss, reduced food consumption, and thymic atrophy (likely secondary to stress). At 635 mg/kg/day in males, body weight loss, reduced food consumption, decreased RBC parameters (one male), increased cholesterol and decreased thymus weight were observed.

870.3100 90

- Da y oral toxicity [Crl:CD

- 1(ICR) mice] 48557829 (2010)

Acceptable/guideline Dietary concentrations of 0, 50, 250, 500 or 1000 ppm, equivalent to: 0, 49.4, 247, 495, or 989 mg/kg/day for males and 0, 50.2, 258, 512, or 1008 mg/kg/day for females NOAEL = 495/1008 mg/kg/day [M/F] LOAEL = 989 mg/kg/day [M], based on increased neutrophils and urinary bladder histopathology. A LOAEL in females was not established (>1008 mg/kg/day).Halauxifen

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Classification /Doses Results

870.3100 90

- Day oral toxicity ( F344/DuCrl rats ) 48557828 (2010)

Acceptable/guideline Dietary concentrations equivalent to: 0, 10.5, 52.4, 262, or 782 mg/kg/day for males and 0, 10.1, 50.3, 252, or 758 mg/kg/day NOAEL = 262/252 mg/kg/day [M/F] LOAEL = 782/758 mg/kg/day [M/F], based o n decreased body weight and body weight gains (males only) , increased urinary volume, decreased urine specific gravity and renal histopathology (necrosis, increased mitotic figures, hypertrophy of collecting duct epithelial cells, vacuolization of collecti ng duct epithelium in the papilla of the kidney, tubular dilatation and degeneration) and increased amorphous crystals in the urine sediment (females only)

.
870.3150 90

- Day oral toxicity (Beagle dog) 48557831 (2011)

Acceptable/guideline Dietary concentrations of 0, 500, 2500 or 12,500 ppm, equivalent to: 0, 16.75, 80.64 or 423.81mg/kg/day in males 0, 18.71, 79.88 or 414.74 mg/kg/day in females NOAEL = 80.64/79.88 mg/kg/day [M/F] LOAEL = 423.81/414.74 mg/kg/day [M/F], based on decreased body weights, decreased body weight gains, reduced food consumption (females), reduced red cell parameters (red blood cells, hemoglobin, hematocrit levels) and additional hematological effects, decreased th ymus weights, thymic atrophy, renal toxicity (including degeneration and regeneration of renal tubules) and extramedullary hematopoiesis of liver and spleen (females).

870.3200 2 8

- Day dermal toxicity ( F344/DuCrl rat s ) 48557834 (2010 )

Acceptable/guideline 0, 100, 3 00, or 1000 mg/kg/day for 6 hrs/day, 7 days/week, 2 8 days Systemic NOAEL = 1000 mg/kg/day Systemic LOAEL = not observed Dermal NOAEL = 1000 mg/kg/day Dermal LOAEL = not observed

Developmental and Reproduction Studies 870.3700a Prenatal developmental [Crl:CD(SD) rats ] 48557835 (2010)

Acceptable/guideline Dietary concentrations of 0, 500, 200 or 8000 ppm, equivalent to 0, 34.1, 140 or 526 mg/kg/day Administered GD 6 through 21 Maternal NOAEL = 140 mg/kg/day LOAEL = 526 mg/kg/day based on increased mortality (moribund sacrifice), decreased fecal output and increased perinasal red soiling, decreased gravid uterine weight, and increased relative kidney weights

. Developmental NOAEL = 140 mg/kg/day LOAEL = 526 mg/kg/day based on decreased fetal weights and increased incidence of delayed ossification of thoracic centra.Halauxifen

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Classification /Doses Results

870.3700b Prenatal developmental probe (NZW rabbit) 48557838 ( 20 1 1 )

Acceptable/ non guideline Dietary concentrations of 0, 5000, 10,000, 15,000 or 23,000 ppm, equivalent to 0, 196, 358, 495 or 929 mg/kg/day Administered GD 7 through 28 Materna l NOAEL = 929 mg/kg/day LOAEL = No dose related effects were seen, thus the maternal LOAEL was not dete rmined. Developmenta l NOAEL = 929 mg/kg/day LOAEL = No dose related effects were seen, thus the developmental LOAEL was not determined.
870.3700b Prenatal developmental (NZW rabbit) 48557837 (2011)

Acceptable/guideline Dietary concentrations of 0, 4000, 10,000 or 28292 ppm, Equivalent to 0, 170, 434 or 1094 mg/kg/day Administered GD 7 through 28 Materna l NOAEL = 434 mg/kg/day LOAEL = 1094 mg/kg/day based on marginally decreased body weight gains and food consumption during gestation

. Developmenta l NOAEL = 1094 mg/kg/day LOAEL = No dose related effects were seen, thus the developmental LOAEL was not determined. 870.3800 Reproduction and fertility effects probe [Crl:CD(SD) rats ] 48557880 (2010)

Acceptable/nonguideline Dietary concentrations formulated to provide doses equivalent to (premating values): 0, 53.4, 255 or 754 mg/kg/day in males and 0, 53.9, 269 or 535 mg/kg/day in females A group administered a target dose of 1000 mg/kg/day was termi nated on Day 8 due to excessive weight loss in some animals. The targeted dose of 750 mg/kg/day for both sexes was reduced to 500 mg/kg/day for both sexes due to excessive weight loss. Parental/Systemic NOAEL = 255/269 mg/kg/day [M/F] LOAEL = 754/535 mg /kg/day based on decreased gestation body weight and weight gain, kidney histopathology (multifocal hypertrophy/hyperplasia of collecting duct epithelial cell lining, nuclear karyomegally, necrosis of individual collecting duct epithelial cells, increased number of mitotic figures within collecting duct cells and tubule dilatation with inflammation) Reproductive NOAEL = 754/536 mg/kg/day [M/F] LOAEL = No dose related effects were seen, thus the reproductive LOAEL was not determined. Offspring NOAEL = 7 54/536 mg/kg/day LOAEL = No dose related effects were seen, thus the offspring LOAEL was not determined.Halauxifen

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Classification /Doses Results

870.3800 Reproduction and fertility effects [Crl:CD(SD) rats ] 48557841 ( 2011 )

Acceptable/guideline Dietary concentrations formulated to provide: P generation premating: 0, 20.7, 104 or 465 mg/kg/day in males and 0, 20.5, 103 or 465 mg/kg/day in females.
F 1 generation: 0, 20.3, 101 or 459 mg/kg/day in males and 0, 19.5, 97.7 or 443 mg/kg/day in females

. Parental/Systemic NOAEL = 104/103 mg/kg/day [M/F] LOAEL = 465/443 mg/kg/day [M/F] based on renal histopathological observations (collecting duct epithelial cell hypertrophy and hyperplasia, hyperplasia of pelvic epithelium, mitotic figures of the collecting duct epithelium) in both sexes ; clinical signs, decreased body weight gains and food consumption during late gestation/early lactation, increased kidney weights, increased incidences of mineralization of the aorta, renal arteries, renal tubular basal lamina and mammary gland artery in females. Reproductive NOAEL = 465/443 mg/kg/day [M/F] LOAEL = No dose related effects were seen, thus the reproductive LOAEL was not determined. Offspring
NOAEL = 465/443 mg/kg/day [M/F] LOAEL = No dose related effects were seen, thus the offspring L OAEL was not determined. Chronic Toxicity Studies 870.4100b Chronic toxicity (Beagle dog) 48557832 (2012)

Acceptable/guideline Dietary concentrations of 0, 5 00, 25 00 or 10,00 0 ppm , equivalent to: 13.90, 82.42, and 354.75 mg/kg/day in males and 16.72, 89.85 and 219.80 mg/kg/day in females NOAEL = 82.42/16.72 mg/kg/day [M/F] LOAEL = 354.75/89.85 mg/kg/day [M/F], based on degeneration with regeneration of the renal proximal and distal tubules, degen eration of collecting ducts , multifocal glomerulosclerosis and in males only, granulomas and fibrosis in the renal cortex

. At 219.80 in females, transient body weight decrease was also observed. Toxicokinetic evaluation: XDE

- 729 showed supralinear kinetic s in plasma of males at 10,000 ppm at 13 and 26 weeks; at 52 weeks, there were insufficient quantifiable samples to assess linearity. Quantifiable levels were not available at terminal sacrifice (fasting), indicating a short half

- life. Most of the dose w as excreted intact in urine; levels were sublinear (not significant) in males at 10,000. Chronic/Carcinogenicity StudiesHalauxifen

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Classification /Doses Results

870.4200b Carcinogenicity [ Crl: CD

- 1(ICR) mice] 48557843 (2012)

Acceptable/guideline Dietary concentrations of 0, 50, 2 50 or 75 0 (males only) or 1000 (females only) ppm , equivalent to: 0, 50, 251, or 751 mg/kg/day for males, and 0, 50.1, 251, or 1004 mg/kg/day for females

. NOAEL = 50 mg/kg/day, both sexes LOAEL = 251 mg/kg/day (both sexes), based treatment

- related subacute to chronic inflammation and microscopic calculi in the lumen of the kidneys of males and decreased body weight gains and increased hypertrophy of the intercalated cells in kidneys of females. At 751 mg/kg/day in males, treatment

- related increases in mortalit y due to renal and urinary bladder lesions , decreased kidney weight, urinary bladder toxicity (thickened bladder wall, hyperplasia of the transitional epithelium lining of the mucosa, increased numbers of mitotic figures within the epithelium, ulceration o f the epithelium, necrosis of individual transitional epithelial cells; decreased aggregates of mononuclear cells secondary to inflammation) and renal toxicity (degeneration with regeneration of cortical tubular epithelium, tubular dilatation, hypertrophy of the intercalated cells of the collecting ducts) were also observed

. N o evidence of carcinogenicity 870.4300 Chronic/ Carcinogenicity ( F344/DuCrl rat s ) 48557842 (2012)

Acceptable/guideline Dietary concentrations formulated to provide dose equivalent to: 0, 20.2, 101, 404 and 633 mg/kg/day , males and 0, 20.3, 102 , 407 or 750 mg/kg/day , females. NOAEL = 101/20.3 mg/kg/day [M/F] LOAEL = 404/101 mg/kg/day [M/F] based on effects in males of increase d mortality , increased urinary volume, decreased urine specific gravity, decreased terminal body weights, increased kidney weights, renal histopathology, hypertrophy of the zona glomerulosa of the adrenal glands, presence of calculi in the renal pelvis of the kidney, degeneration and regeneration of renal tubu les and bladder his topathology (including calculi); and in females, based on hyperplasia of pelvic epithelium of the kidney. At 407 mg/kg/day in females, increases in chronic progressive glomerulonephropathy (12 months only) and chronic interstitial infla mmation were noted in the kidneys

. At 633 mg/kg/ day, increased mortality was observed in males. Body weight/weight gain were decreased in both sexes. Females given 750 mg/kg/day were terminated on test day 518 due to excessive mortality (56%) and body weight , indicat ing exceeding the maximum tolerated dose (MTD); these animals were subsequently removed from the study and no histopathological assessments were conducted.
N o evidence of carcinogenicityHalauxifen

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Classification /Doses Results
Mutagenicity Studies Gene Mutation 870.5100 Bacterial Reverse Mutation Test (Pre

incubation assay) 48557844 ( 2010 )

Acceptable/guideline 0, 50, 10, 500, 1 5 00, or 5000 μg/plate +/

- S9 Negative in Salmonella typhimurium strains TA 1535, TA1537 , TA98, and TA100 and Esc h erichia coli Wp2 uvrA up to the limit concentration with no evidence of compound insolubility or cytotoxicity

.

Gene Mutation 870.5300 In vitro mammalian cell forward gene mutation, Chinese hamster ovary (CHO) cells , HGPRT locus

48557848 (2010) Acceptable/guideline 0, 187.5, 375, 750, 1500, 2000, 2500, 3000 or 3400 μg/plate +/

- S9 Negative for induced mutant colonies at the HGPRT locus up to precipitating and cytotoxic concentrations.

Cytogenetics 870.53 75 In vitro mammalian cell chromosomal ab erration assay, primary lymphocytes (Sprague

- Dawley rats) 48557852 ( 2010 )

Acceptable/guideline 0, 800 , 15 00 or 2000 μg/mL in the absence of mammalian metabolic activation (S9) ; 0, 40 0, 800 or 1500 μg/mL in the absence of mammalian metabolic activation Negative for induction of chromosomal aberrations up to cytotoxic concentrations.

870.5395 Cytogenetics In vivo mammalian micronucleus assay [Crl:CD1(ICR) mice] 48557856 (2010)

Acceptable/guideline 0, 500, 1000 or 2000 mg/kg Negative for inc reased frequency of micronucleated reticulocytes in peripheral blood up to a dose causing systemic toxicity.

Neurotoxicity Studies
870.6200a Acute Neurotoxicity Screening Battery ( F344/DuCrl rats) 4 85578 5 7 (20 10)

Acceptable/guideline 0, 250 , 7 5 0 , or 200 0 mg/kg (gavage) Neurotoxicity NOAEL = 2000 mg/kg (HDT) Neurotoxicity LOAEL not established (>200 0 mg/kg ) Systemic NOAEL = 200 0 mg/kg Systemic LOAEL not established (> 2000 mg/kg ) ,
870.6200b Subchronic Neurotoxicity Study ( F344/DuCrl rats) 4 8557858 (20 11 )

Acceptable/guideline Dietary concentrations equivalent to 0, 5 0, 250, or 7 5 0 mg/kg/day Neurotoxicity NOAEL = 750 mg/kg/day (HDT) Neurotoxicity LOAEL not established (>750 mg/kg/day) Systemic NOAEL = 7 50 mg/kg /day Systemic LOAEL not established (> 750 mg/kg /day) ,
Other StudiesHalauxifen

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Classification /Doses Results

870.7485 Metabolism and pharmacokinetics probe study [ F344/DuCrl rats) and Crl:CD1(ICR) mice] 4 8557859 ( 2009 )

Acceptable/ non guideline 100 mg/kg single dose (gavage) radiolabeled XDE

- 729 acid, [Phenyl

- U

14 C] label XDE

- 729 acid was rapidly and well

- absorbed from the GI tract at comparable rates for mice and rats. Elimination was primarily via the urine (65

- 75% AD in rats and 65

- 66% AD in mice). Fecal elimination accounted for 12

- 21% AD in rats and 19

- 20% AD in mice. Radioactivity was not found in expired air. Kinetics was comparable for male and females. Peak plasma concentrations (C max ) was achieved at 0.5 hr in both species, plasma half

- life (T 1/2α ) was 0.14 hr and terminal elimination half

- life (T 1/2ß ) was 3.37 hr. AUC was 529 μg/h/g

- 1 in males and 428 μg/h/g

- 1 in females. In both species and in males and females, XDE

- 729 was mainly excreted as unchanged parent compound, with six minor peaks observed in urine. Of these, glucuronide and sulfate conjugates of O

- d emethyl XDE

- 729, O

- demethyl XDE

- 729 and an acyl

glucuronide conjugate were identified. Parent XDE

729 was also the major radiolabeled component in rat and mouse plasma at C max. Total recovery was 89

- 90% in rats and 92

- 93% in mice.Halauxifen

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Classification /Doses Results

870.7485 Metabolism and pharmacokinetics [ F344/DuCrl rats) 48557860 (2010)

Acceptable/guideline radiolabeled XDE

- 729 acid, sodium salt [Phenyl

14 C] label: Single oral dose, 10 mg/kg; single oral dose, 750 mg/kg; 14

- day oral repeat dose, 10 mg/kg/day; IV dose, 10 mg/kg; radiolabeled XDE

- 729 acid, sodium salt [P yridine

14 C] label: Single oral dose, 10 mg/kg; radiolabeled XDE

- 729 methyl ester, [Phenyl

- U

14 C] label: 10 mg/kg single gavage dose, 10 mg/kg XDE

- 729 sodium salt was rapidly and well

- absorbed from the GI tract (at least 68

- 92% based on recovery in urine and non

- GI tissues). Relative oral bioavailability was about 100% based on comparison of the areas under the curve of the oral and IV 10 mg/kg dose groups. Kinetics and metabolite profiles were similar for males and females. Maximum plasma concentration was achieved in 0.2 hr. The initial elimination half

- life at 10 mg/kg (T 1/ 2α ) was 0.5

- 0.9 hr, followed by a slower phase (T 1/2ß ) of 6

- 16 hr. The initial elimination phase at 750 mg/kg was slower (2.7

- 4.3 hr) but not the terminal phase (5.6

- 8.7 hr). The AUC for the high dose was 2.0

- 2.6 times higher than would be expected for dose proportionality. Kinetics of radioactivity in RBC were less than plasma. Elimination was primarily via the urine (68

- 92% AD), with some excretion in the feces (11

- 29% AD). Excretion was rapid, with most of the oral dose excreted within 24 hr. Biliary excretion was observed, based on fecal excretion of 11

- 15% AD after IV dosing. Radioactivity in tissues was very low, with only 0.3% and 0.03% AD remaining in tissues by 168 hours for the oral and IV low dose, respectively. The highest levels were generally found in the skin or GI tract. XDE

- 729 sodium salt was poorly metabolized and primarily excreted as unchanged parent. Minor metabolites of consisted of the acyl

- glucuronide conjugate of parent XDE

- 729 (present in selected samples at greater than 5% of the administered dose) and O

- demethylated XDE

- 729 and the corresponding sulfate and glucuronide conjugates . Recovery was close to 100% for all groups. XDE

- 729 methyl: The absorption, tissue distribution, elimination kinetics and metabolite profiles were similar to XDE

- 729 sodium salt, indicating rapid hydr olysis of the methyl ester in vivo , resulting in bioequivalence. Peak plasma concentration was achieved by 0.4

- 0.5 hr. Blood samples collected at or near peak showed that the major component was XDE

729 acid. XDE

- methyl showed an increased compared to X DE

- 729 sodium salt, but AUCs were the same.Halauxifen

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Classification /Doses Results

870.7485 Metabolism and pharmacokinetics probe study (Beagle dog) 48557870 (2012)

Acceptable/nonguideline 50 mg/kg single dose (gavage) radiolabeled XDE

- 729 acid, sodium salt [Phenyl

- U

14 C] label XDE

- 729 acid was rapidly and well

- absorbed from the GI tract in male and female beagle dogs. Plasma C max was achieved at 0.7 hr in females (143 μg

- eq./g) and 1.3 hr in males (118 μg

- eq./g). Elimination from the blood was biphasic, with an initial half

- life (T 1/2α ) of 1 hr and terminal half

- life (T 1/2ß ) of 9

- 12 hr. Excretion was primarily via the urine (about 80% AD), mostly within 12 hr, with about 10

- 14% excreted in feces, mostly by 48 hr. XDE

- 729 was excreted primarily as unchanged parent compound. Minor metabolit es in urine, feces or plasma included O

- demethyl XDE

- 729 and the sulfate and glucose conjugates of O

- demethyl XDE

- 729, hydroxy XDE

- 729, and an acyl glucuronide conjugate of XDE

- 729. Total recovery by 168 hr was 95

- 98% AD. Non

- Guideline Studies See XDE

- 729 methyl for Mechanism of Action (MOA) studies for liver effects

Subchronic, Chronic, and Other Toxicity Studies for Metabolite X11449757 Guideline No./ Study Type MRID No. (year)/

Classification /Doses Results

Mutagenicity Studies Gene Mutation 870.5100 Bacterial Reverse Mutation Test (Pre

incubation assay) 48557847 ( 2012 )

Acceptable/guideline 0, 156.25, 312.5, 625, 1250, 2500 or 5000 μg/plate

- S9 activation and 0, 51.2 , 1 28 , 32 0, 8 00, 2000 or 5000 μg/plate + S9 activation Negative in Salmonella typhimurium strains TA 1535, TA1537 , TA98, TA100 and TA10 2 and Esc h erichia coli Wp2 uvrA up to the limit concentration with no evidence of compound insolubility or cytotoxicity

.

Gene Mutation 870.5300 In vitro mammalian cell forward gene mutation, Chinese hamster ovary (CHO) cells , HGPRT locus

48557855 (2012) Acceptable/guideline 0, 100, 200, 400, 800, 1600 or 3210 μg/ mL +/

- S9 activation Negative for induced mutant colonies over background at the HGPRT locus up to precipitating and cytotoxic concentrations.Halauxifen

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Classification /Doses Results

870.5375 Cytogenetics In vitro mammalian cell chromosomal aberration assay , human peripheral lymphocytes 48557850 (2012)

Acceptable/guideline 0, 25.3, 50.6, 101.3, 202.5, 405 or 810 μg/ mL +/

- S9 activation Negative for increased chromosomal aberrations over background up to cytotoxic and precipitating concentrations.

A.3 Hazard Identification and Endpoint Selection
A.3.1 Acute Reference Dose (aRfD)
- Females age 13
- 49 An appropriate endpoint was not identified for a single exposure to females age 13

- 49. No effects from a single

- dose exposure were identified for either halauxifen

- methyl or XDE

- 729 acid.
A.3.2 Acute Reference Dose (aRfD)
- General Population An appropriate endpoint was not identified for a single exposure to the general population. No effects from a single

- dose exposure were identified for either halauxifen

- methyl or XDE

- 729 acid.
A.3.3 Chronic Reference Dose (cRfD )
Halauxifen
- Methyl:
Stud y Selected: 90

- day oral toxicity in the rat MRID No .: 48557830 Executive Summary : See A ppendix A, Guideline §870.3100a Dose and Endpoint for Risk Assessment: POD = 3.0 mg/kg/day based on increased expression of Cyp1a1 in the live r at 10.3 mg/kg/day. Comments about Study/Endpoint/Uncertainty Factors: The rat 90

- day oral study was selected because long

- term oral toxicity studies are not available for halauxifen

- methyl. The study NOAEL is 10.3 mg/kg/day, based on increased liver weight, hypertrophy and vacuolization consistent with fatty change at the LOAEL of 53.4 mg/kg/day

. L iver effects at the LOAEL were of low severity. A marked increase (1500

- fold above controls) in Cyp1a1 expression was also observed at the LOAEL. Mechanistic studies on halauxifen

- methyl identified activation of liver AhR as the MOA for liver toxicity, f

or which increased expression of Cyp1a1 in the liver is a bio marker for AhR activation, the MIE. Although there were no liver effects at the study NOAEL, a 5 2

- fold increase in Cyp1a1 expression was observed. Long

- term effects on the liver from this lower level increase are not known in the absence of chronic data. Therefore, in order to be protective of potential adverse effects on the liver from lower

- level increases in Cyp1a1 expression at longer exposureHalauxifen

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Page 64 of 126 durations, the POD of 3 mg/kg/day was selected , based on increased expression of liver Cyp1a1 (5 2

- fold) at 10 mg/kg/day. Cyp1a1 expression at 3 mg/kg/day was comparable to controls in both the 28

- and 90

- day studies (1.2

- and 3.6

- fold higher than controls, respectively), indicating that there is not e xpected to be a biologically significant activation of the AhR receptor at this dose level over time. The dose and endpoint are considered conservative, but protective of residual uncertainty due to the lack of chronic data, and are also protective of chr onic effects from XDE

729 acid, which are observed at higher doses. A UF of 100 is based on the combined interspecies (10x) and intraspecies (10x) UFs. An additional 10x UF s for lack of chronic data was not applied for the following reasons: (1) progres sion of toxicity was not observed in the 28

- and 90

- day dietary studies in the rat: the NOAELs and LOAELs for both studies were the same, and the severity of the findings was minimal at both exposure durations; (2) evaluation of Cyp1a1 expression in the r at 7, 28 and 90

- day studies indicated that at the selected POD, which is below the NOAELs for these studies, there is no expectation of significant AhR activation that c ould lead to liver toxicity; (3) halauxifen

- methyl is rapidly metabolized to the acid, and neither bioaccumulate; (4) based on in vitro studies, humans are not anticipated to be more sensitive to liver effects of halauxifen

- methyl, and (5) dietary exposure to humans is expected to be very low (<1% of the cPAD) due to the low application rate s and environmental degradation.
XDE
- 729 acid:
Stud y Selected: Chronic toxicity/carcinogenicity in the rat MRID No s .: 48557842 (rat) Executive Summary : See A ppendix A, Guidelines §870.4300 (rat) Dose and Endpoint for Risk Assessment: NOAEL = 20.3 mg/kg/day based on hyperplasia of the renal pelvic epithelium in females at 101 mg/kg/day. Comments about Study/Endpoint/Uncertainty Factors: The selected study is of the appropriate route and duration for assessment of chronic dietary exposure. A si milar NOAEL and LOAEL of 16.7 and 89.9 mg/kg/day, respectively, based on renal tubule toxicity in females, were identified in the one year oral dog study: the rat study is considered protective based on comparison of dose

- spacing in the two studies. The NOAEL of 20.3 is protective of developmental effects, observed in the rat at 526 mg/kg/day (NOAEL = 140 mg/kg/day) , and of maternal toxicity in both the rat (LOAEL = 526 mg/kg/day) and rabbit (LOAEL 1094 mg/kg/day). Parental toxicity in the rat two

- gener ation reproductive toxicity study was observed at 443 mg/kg/day (NOAEL 103 mg/kg/day), but no offspring or reproductive toxicity was reported. A UF of 100 is based on the combined interspecies (10x) and intraspecies (10x) UFs.
A.3.4 Incidental Oral Ex posure (Short
- and Intermediate
- Term) Residential post

- application exposure is not anticipated because there are no proposed residential uses. An endpoint was therefore not selected at this time.
A.3.5 Dermal Exposure (Short
- , Intermediate
- and Long
- Ter m )

– Halauxifen
- methyl An appropriate dose and endpoint for dermal exposure were not identified. In a rat 28

- day dermal toxicity on XDE

- 729 acid, no toxicity was observed. However, occupational exposure will be to halauxifen

- methyl in end

- use products. Although a dermal toxicity study onHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 65 of 126 halauxifen

- methyl was not conducted, dermal absorption data on halauxifen

- methyl are available. In vivo absorption of halauxifen

- methyl in the rat was 24% of the applied dose, with toxicokinetic data showing that XDE

- 7 29 acid, but not halauxifen

- methyl, was present in the blood, indicating rapid hydrolysis following absorption. Application of the DAF of 24% to the LOAELs from rat studies on XDE

- 729 acid of the most sensitive rat studies of the appropriate duration (rat developmental, rat two

- generation reproductive toxicity) indicated that toxicity would be expected at doses approximately twice the dermal limit dose of 1000 mg/kg/day, and adjusted NOAELs were 410

- 640 mg/kg/day. Therefore, since toxicity from dermal exp osure would only be expected at exposures far exceeding potential human dermal exposure, an endpoint was not selected. A 28

- or 90

- day dermal toxicity study on halauxifen

- methyl was therefore not needed (HASPOC memorandum, TXR No. 0056954).
A.3.6 Inhala tion Exposure (Short
- and Intermediate
- Term)

– Halauxifen
- methyl Study Selected: 90

- day oral toxicity in the rat, halauxifen

- methyl MRID No.: 48557830 Executive Summary : See Appendix A, Guideline §870. 3100a Dose and Endpoint for Risk Assessment: POD = 3.0 mg/kg/day based on increased expression of liver Cyp1a1 at 10 mg/kg/day

. Comments about Study/Endpoint/Uncertainty Factors: Inhalation exposure will be to halauxifen

- methyl in end

- use products. N o inhalation studies are available

. Therefore an oral study, the rat 90

- day toxicity study on halauxifen

- methyl, was selected for assessment of inhalation exposure. The selected oral study is of an appro priate exposure duration for assessment of short

- and intermediate

- term exposure risk. T he study NOAEL was 10.3 mg/kg/day, based on slightly increased liver weight, hepatocellular vacuolization and cholesterol at the LOAEL of 53 mg/kg/day. At the LOAEL, a marked increase in Cyp1a1 expression was observed (1500

- fold) but a much lower increase (5 2

- fold) was also seen at the NOAEL of 10.3 mg/kg/day. Therefore, in the absence of information on the toxicity of halauxifen

- methyl to the respiratory tract, a POD of 3 mg/kg/day, based on the endpoint of increased Cyp1a1 expression in the liver at 10 mg/kg/day, was selected. The selected POD is the most sensitive dose available and is protective of AhR activation , an adverse outcome pathway that may occur in the lung as well as the liver . The dose is also protective of maternal toxicity in the rabbit , the more sensitive species of the developmental studies (NOAEL of 5.8 mg/kg/day, based on liver enlargement and hepatocellular histopathology at the maternal LOAEL of 18.5 mg/kg/day) as well as developmental toxicity (decreased fetal body weights and del ayed ossification, observed at 72 mg/kg/day). Toxicity via the inhalation route is assumed to equivalent to toxicity via the oral route . The UF of 100x is based on the combined interspecies (10x) and intraspecies (10x) UFs. An additional UF DB was not included for lack of inhalation exposure data because the selected dose is conservative, is below the NOAEL for adverse effects in the rat oral 90

- day study and is protective of AhR activation/Cyp1a1 induction . It is also noted that halauxifen

- me thyl has a low vapor pressure ( 4.4 x 10 - 11 mmHg at 25°C) , reducing the likelihood of significant inhalation exposure. MOEs for inhalation exposure calculated using the oral POD were adequately protective (>1000). In addition, HED has determined that a ro ute

- specific inhalation study is not needed for halauxifen

- methyl.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 66 of 12 6 A. 4 Executive Summaries
Halauxifen
- Methyl (XDE
- 729 Methyl)
A.4.1 Subchronic Toxicity
870.3050 28
- Day Oral Toxicity

– Rat
In a subchronic oral toxicity study (MRID 48557826, PMRA 2226461) , f ive F344/DuCrl rats /sex/ group were at test diets formulated to supply 0, 10, 52, 261, or 782 mg XDE

- 729 methyl (97.2% a.i., lot no

. E2837

- 51, TSN031117

- 0004 ) / kg body weight/day for 30 days. Average intake of XDE

- 729 methyl corresponded to time

- weighted average doses of 0, 10.8, 55.6, 296, or 822 mg/kg bw /day for males and 0, 10.7, 55.5, 277, or 825 mg/kg bw /day for females, respectively. In addition to standard toxicity parameters, blood (three time points) and urine sampl es (overnight collection) were collected during the fourth week of treatment and toxicokinetic analysis was performed.
There were no treatment

- related effects on survival, clinical signs, urinalysis parameters or gross pathology. Treatment

- related decrea ses in body weight gains (11

- 17%) and food consumption (7

- 20%) were observed at ≥277 mg/kg bw /day in females. Hematological effects consisted of decreases in red blood cell count, hematocrit, and/or hemoglobin concentration (2

- 6% each) in males and females at 8 22 /82 5 mg/kg bw /day ; these changes were considered to be treatment

related but non

- adverse by Australia OCS . These alterations corresponded with slight erythroid cell hyperplasia of the bone marrow (incidences: ♂ 1, 1, 0, 0, 4; ♀ 0, 1, 1, 0, 2) and increased erythrocytic extramedullary hematopoiesis of the spleen (incidences: ♂ 1, 1, 0, 0, 4; ♀ 1, 1, 3, 1, 4) . Clinical chemistry effects consisted of higher cholesterol concentrations in males and females at ≥277 mg/kg bw /day (♂ 50

- 115%, ♀ 83

- 95%) and higher aspartate aminotransferase (AST) activities (♂ 35%, ♀ 24%) in high

- dose males and females.
Treatment

- related statistically identified increases in the mean absolute and/or relative liver weights (absolute/relative ♂ 7

- 41/8

- 46% , ♀ 13

- 18/8

- 29%) were noted in males and females at 5 5.6/55.5 mg/kg bw /day M/F relative to controls. In males at ≥55.6 mg/kg bw/day, there was associated hepatocellular hypertrophy (incidences: 0, 0, 3, 5, 5), increased mitotic figures in hepatocytes (inc idences: 0, 0, 1, 3, 4) and hepatocellular vacuolization (consistent with fatty change) (incidences: ♂ 0, 0, 3, 4, 5). In females, the first two liver histopathological changes were observed at 277 and 825 mg/kg bw /day (incidences: ♀ 0, 0, 0, 5, 5 and 0, 0 , 0, 0, 1 respectively). The other primary histopathological effect was hypertrophy of collecting duct epithelial cells in the kidneys of two high

- dose males.
Males at 296 and 822 mg/kg bw /day had treatment

- related higher absolute (16

- 29%) and relative ( 15

- 33%) thyroid weights , which were considered to be toxicologically relevant as this change was associated with treatment

- related hypertrophy of thyroid gland follicular cells at ≥ 55. 6 mg/kg bw /day (incidences: 0, 0, 2, 5, 5) . Males and females at 822/825 mg/kg bw /day , respectively, had treatment

- related lower absolute (♂ 17%, ♀ 14

- 21%) and relative (♂ 15%, ♀ 10

- 14%) thymus weights. Males and females at 825 mg/kg bw /day had treatment

- related acinarHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 67 of 126 cell hypertrophy of the submandibular salivary gland (inci dences: ♂ 0, 0, 0, 0, 1; ♀ 0, 0, 0, 0, 4) and lymphoid atrophy of the thymus. The thymic lymphoid atrophy was a minimal effect that was only present in 2 males and 2 females from the high

- dose group. Treatment

- related decreases in the size of the cervix, uterus and vagina were observed in high

- dose females (incidences: 2, 1, 2 vs. 0 in controls, respectively)

. The male LOAEL is 55.6 mg/kg bw /day, based on increased liver weight, hepatocellular hypertrophy, increased mitotic figures in hepatocytes, hepatocellular vacuolization (consistent with fatty change) and hypertrophy of thyroid gland follicular cells. The female LOAEL is 277 mg/kg bw /day, based on decreased body weight gains and food consumption, increased liver weight, increased cholesterol le vels and hepatocellular hypertrophy. The NOAEL in males is 10.8 mg/kg bw /day and the NOAEL in females is 55.5 mg/kg bw /day. After 4 weeks of exposure to XDE

- 729

- methyl in the diet, XDE

- 729

- methyl was not present at detectable levels in the whole blood of most male and female rats, except at the highest dose in males at low levels . However, XDE

- 729 acid was present in blood samples from both sexes at all four doses and almost all time points. Both XDE

- 729 acid and XDE

- 729

- methyl were present in the urine of male and female rats collected over 24 hours on the same day as the blood sample collection. Less than 0.066% of the administered dose was excreted as XDE

- 729

- methyl in the 24 hour urine, and 23

- 43% of the administered dose was excreted as the metabolit e XDE

- 729 acid

. Daily systemic exposures (AUC 24h ) for XDE

- 729 acid and the total amounts excreted in the 24 hour urine were dose

- proportional (linear) at all doses in males, but only up to the mid

- high dose in females. The daily systemic exposures for X DE

- 729 acid and the total amount of XDE

- 729 acid and XDE

- 729

- methyl excreted over 24 hours in urine in females of the high

- dose group were both less than dose

- proportional (sublinear), suggesting possible saturation of absorption and/or metabolism at the h igh dose in female rats.
This subchronic toxicity study in the rat is classified as acceptable /guideline and satisfies the guideline requirement (OECD 407, OPPTS 870.3050) for a 28

- day repeat

- dose oral study in rats.
870.3100 90
- Day Oral Toxicity

– Rat
In a subchronic oral toxicity study (MRIDs 48557830, amended report 49124405; PMRA 2315015) , XDE

- 729 Methyl ( 97.2% a.i., lot no. E2837

- 51; TSN031117

- 0004) was administered to 10 DuCrl/F344 rats /sex/g roup in test diets formulated to supply 0, 3, 10, 52, 26 1, or 500 m g/kg bw/day for at least 90 days . The actual doses to the animals equaled 0, 3.1, 10.3, 53.4, 270 or 513 mg/kg bw/day in males and 0, 3.0, 10.1, 52.3, 263 or 504 mg/kg bw/day in females. In addition to standard guideline parameters, males were evaluated for liver gene expression and toxicokinetic analysis of the test material in liver, blood and urine. There were no treatment

- related effects o n survival, clinical signs, functional tests, ophthalmic parameters , f

oo d consumption, thyroid hormones, urinalysis parameters or gross pathologic observations.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 68 of 126 Treatment

- related decreases in body weights (♂ 5%, ♀ 7%) and body weight gains (♂ 8%, ♀ 14%) were observed in high

- dose males and females throughout the study but were not considered of sufficient m agnitude to be of toxicological significance.
F emales at 26 3 and 5 04 mg/kg bw /day had lower percent neutrophils (27

- 31%) and higher percent lymphocytes (7

- 8%) , relative to controls that were interpreted to be treatment

- related. Males at ≥ 270 mg/kg bw/day and females at ≥ 52.3 mg/kg/day had treatment

- related higher cholesterol concentrations (♂ 41

- 73%, ♀ 24

- 59%). Males and females at 270/263 or 513/504 mg/kg bw/day M/F had treatment

- related lower alkaline phosphatase activities (♂ 21

- 25%, ♀ 14

- 20%) and high

- dose males had lower aspartate aminotransferase activities (18%). Males at 270 or 513 mg/kg bw/day and females at 52.3, 263, or 504 mg/kg bw/day had treatment

- related increases in absolute and relative liver weights (absolute /relative ♂ 22

- 34%/22

- 40%, ♀ 12

- 26%/12

- 36%), which were associated with panlobular hepatocellular hypertrophy with altered tinctorial properties (increased cytoplastic eosinophilia) in both sexes at the two highest dose levels in all animals. Other treatme nt

- related liver effects consisted of increased numbers of mitotic figures (hepatocytes in mitosis) in females at 263 or 504 mg/kg bw/day (incidences: 0, 0, 0, 0, 2, 5), and hepatocellular vacuolization (consistent with fatty change) in males at ≥ 52.3 mg/ kg bw/day and females at ≥ 263 mg/kg bw/day (incidences: ♂ 0, 0, 0, 5, 10, 10; ♀ 0, 0, 0, 0, 8, 5). A subsequent 7

- day mode

- of

- action (MOA) study was performed in male F344 rats given a similar high dose of XDE

- 729 methyl or XDE

- 729 (acid) to compare live r gene expression responses for Cyp1a1 , Cyp2b1 , Cyp3a23 , and Cyp4a22 as biomarkers for activation of the AhR, CAR, PXR, and PPAR

-  signaling pathways, respectively. Results from this study indicated that XDE

- 729 methyl, but not XDE

- 729 (acid), was activat ing the AhR signaling pathway as indicated by a robust induction in Cyp1a1 transcript levels. The appearance of histopathological apical effects in the liver at ≥ 53.4/52.3 mg/kg bw /day M/F correlated with a dose

- responsive induction in Cyp1a1 and Ugt1a6 g ene expression, which is consistent with activation of the AhR signaling pathway. Transcript levels for Cyp1a1 demonstrated the highest induction of 1495 and 2268

- fold at 53.4/52.3 mg/kg bw/day in males and females, respectively

. In addition, levels of XD E

- 729 methyl in the liver were increasing in a dose responsive manner across these treatment groups. Induction of Cyp1a1 transcript levels occurred in male and female livers in a dose

- responsive manner at 5 3.4/5 2 .3 mg/kg/day and above M/F . Therefore, inc reases in Cyp1a1 transcript levels of greater than 1000

- fold were associated with the appearance of histopathological and apical effects in the liver at 53.4/ 52 .3 mg/kg bw /day M/F

. At the two highest doses , further increases in Cyp1a1 transcript levels were observed which were associated with increasing liver weights and a greater severity in the histopathological responses. In addition to liver effects, t he thyroid hypertrophy observed in males and females at ≥ 270/ 26 3 mg/kg bw /day M/ F was associated with a concomitant dose responsive induction of liver Ugt1a6 transcript levels. Males and females at 270/263 or 513/504 mg/kg bw/day M/F had treatment

- related increased absolute and relative thyroid weights (absolute/relative ♂ 14

- 23%/13

- 28%; ♀19

- 25%/26

- 28%), which corresponded to microscopic thyroid follicular cell hypertrophy (incidences: ♂ 1, 0, 0, 1, 9, 9; ♀ 0, 0, 1, 0, 8, 10). The thyroid hypertrophy observed in males and females at the two highest dose levels was associated with a dose responsive induction of liver Ugt1a6 transcript levels. There were no treatment

- related effects on thyroid hormone levels at necropsy; however,Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 69 of 126 the sampling time likely occurred after a compensatory mechanism by the hypothalamic

pituitary

- thyroid (HPT ) axis. The high

- dose females had lower absolute and relative thymus weights (absolute/relative 30%/24%), with corresponding atrophy of thymic lymphoid tissue (incidences: 0, 0, 0, 1, 0, 6). These changes were considered to be treatment

- related.
The LOAE L in males is 53.4 mg/kg bw/day, based on increased hepatocellular vacuolation (consistent with fatty change) and increased Cyp1a1 gene expression. The LOAEL in females is 52.3 mg/kg bw/day, based on increased liver weights and increased cholesterol levels and increased Cyp1a1 gene expression. The NOAEL in males and females is 10.3 and 10.1 mg/kg bw/day, respectively. However, it is noted that a mild increase in the induction of Cyp1a1 gene expression (52

- and 149

- fold above controls, males and females) wa s observed at the study NOAEL of 10.3 mg/kg bw/day. At 3.1 mg/kg bw/day, there was only a slight increase (3.6

- and 5.7

- fold, males and females). Toxicokinetic analysis results indicated that the parent, XDE

- 729 methyl, was present at quantifiable levels in the liver (all doses) and the blood (≥10 mg/kg bw/day males; 52 and 500 mg/kg/day females). Consistent with hepatic induction of the AhR signaling pathway by XDE

729 methyl , l evels of XDE

- 729 methyl in the liver were increasing in a dose

- responsive manner across these treatment groups. The primary metabolite, XDE

- 729 acid, was present in blood, liver, and urine samples from both male and female rats at all doses and almost all time

- points. Five additional metabolites were also detected in the blood, liver, and urine of most animals. Th e sulfate conjugate of O

demethylated XDE

- 729 acid appeared to be the most prevalent of these in males, while the glucuronide conjugates of O

- demethyl XDE

- 729 methyl and XDE

- 729 acid were the most prevalent in females. In the liver, XDE

- 729 methyl was qu antifiable at low levels from all treated animals and exhibited kinetic linearity. However, the primary metabolite, XDE

- 729 acid, was the most abundant and exhibited sublinearity in males at ≥270 mg/kg bw/day. Similarly, the O

- demethyl XDE

- 729 metabolite was sublinear (both sexes) at ≥270/263 mg/kg bw/day M/F, while the glucuronide conjugate of this O

- demethyl XDE

- 729 metabolite exhibited supralinearity in males at these dose levels. In whole blood, XDE

- 729 methyl was not present at quantifiable levels in a majority of males and females at all doses, except at the highest dose level. Similar to the liver samples, the primary metabolite XDE

- 729 acid was sublinear in male blood at ≥270 mg/kg bw/day. Analysis of the urine samples revealed that most of th e administered dose of XDE

- 729 methyl was rapidly metabolized and excreted in the urine. XDE

- 729 methyl was not present above LLQ in urine, except in one high

- dose urine sample. All of the metabolites exhibited supralinear kinetics in urine at ≥53.4/52.3 m g/kg/day M/F. Conversely, XDE

- 729 acid levels decreased with increasing doses in both sexes. These data suggested kinetic nonlinearity in the metabolism and elimination of administered XDE

- 729 methyl, with an increase in metabolic conversion to conjugate d metabolites with increasing doses, which were in turn more rapidly eliminated in theHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 70 of 126 urine. These data are consistent with an increase hepatic induction of metabolism enzymes at ≥5 3.4 / 52.3 M/F mg/kg bw/day.
This subchronic oral toxicity study in the rat s is classified as acceptable /guideline and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats.
870.3050 28
- Day Oral Toxicity
- Mouse Study not conducted for halauxifen

- methyl. See Executive Summaries f or XDE

- 729 Acid
870.3100 90
- Day Oral Toxicity
- Mouse Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid
870.3150 90
- Day Oral Toxicity
- Dog Study not conducted for halauxifen

- methyl: see Executive Summaries for XDE

- 729 acid
870.3200 21/28
- Day Dermal Toxicity

– Rat Study not conducted for halauxifen

- methyl: see Executive Summaries for XDE

- 729 acid
870.3465 90
- Day Inhalation

– Rat Study not conducted due to inability to generate acceptable test atmosphere
A .4.2 Prenatal Developmental Toxicity Nonguideline Prenatal Developmental Rangefinding Study

– Rat
In a n oral range
- finding developmental toxicity study (MRID 48557881; PMRA 2226497) , XDE

- 729 methyl ( 97.2% a.i., lot no. E2837

- 51, TSN031117

- 0004) was administered to 5 time

mated female Crl:CD(SD rats /dose in die t at dose levels of 0, 521, 2083, 4167, or 8333 ppm on gestation day (GD) 6 through 21, (equal to 0, 40.4, 169, 303, or 490 mg/kg bw /day, respectively )

. Limited parameters were examined; det ailed fetal observations were not performed. Blood concentrations of XDE

- 729 methyl and the metabolite XDE

- 729 acid were determined in samples from all rats on GD 20 and 21 and their respective litters at necropsy. The animals in the highest dietary conc entration, 8333 ppm (490 mg/kg bw /day), were sacrificed prematurely based on excessive reductions in body weight gains (90% decrease from controls) from GD 6

- 9 ; this dose was considered to have exceeded the maximum tolerated dose . In the remaining animals at the lower dose levels , there were no treatment

- related clinical signs or effects on body weights, gross pathology, organ weights or Caesarian section parameters. In the 303 mg/kg bw/day maternal animals, treatment

- related decreases in body weight gain s (89%) and food consumption (33%) were observed throughout the study. A detailedHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 71 of 126 developmental assessment was not conducted in this range
- finding study. Blood concentrations showed linear kinetics across all three doses for both XDE

- 729 methyl, when pres ent, and the metabolite XDE

- 729 acid . XDE

- 729 methyl was present above the lower limit of quantification ( LLQ ) during early sampling times on GD 20 and was absent in GD 21 dams. In contrast, the main metabolite, XDE

- 729 was present in all dams and fetal bl ood samples. When present, XDE

- 729 methyl concentrations were ~0.9% that of the metabolite XDE

- 729 acid . Fetal blood concentrations of XDE

- 729 acid were ~69% of the dam blood concentrations. Canada PMRA, Australia OCS: No LOAELs or NOAELs were establish ed in this supplemental range
- finding study. US EPA: Maternal NOAEL and LOAEL were selected for comparison with the definitive study. The maternal LOAEL is 303 mg/kg bw/day, based on decreased food consumption and body weight gains. The maternal NOAEL is 169 mg/kg bw/day. A developmental NOAEL/LOAE L was not determined since a fetal examination was not performed. The developmental toxicity study in the rat is classified as acceptable /non

- guideline for a supplemental dose range

- finding study that was not conducted to satisfy a guideline requirement for a rat developmental toxicity study. 870.3700a Prenatal Developmental Toxicity Study

– Rat In an oral developmental toxicity study ( PMRA 2226499 , MRID 48557836), g roups of 24 time

mated female Crl:CD(SD) rats were administered diets containing 0, 521, 2083, or 4167 ppm XDE

- 729 methyl (97.2% a.i., lot no. E2837, TSN031117

- 0004) on gestation day (GD) 6 through 21 which corresponded to time

- weighted average doses of 0, 41.1, 159, or 323 mg/kg bw /day, respectively. In

- life maternal study parameters included clinical observations, body weight, body weight gain and f

oo d consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetu ses were examined for visceral and craniofacial alterations, while skeletal examinations were conducted on the remaining fetuses. Blood concentrations of XDE

- 729 methyl and t he metabolite XDE

- 729 acid were analyzed in samples collected from four dams/grou p and their respective litters at necropsy. Maternal toxicity : There were no treatment

- related effects on survival, clinical signs, gross pathology or Caesarian section parameters in maternal animals. At 323 mg/kg bw/day, decreased body weight gains (23%) and food consumption (8%) were observed from GD 6

- 9 and were considered to be adverse. Treatment

- related increases in liver weight ( absolute/relative 22%/21%), altered cytoplasmic homogeneity of centrilobular and midzonal hepatocytes (incidences: 2, 3, 14, 21), and hepatocellular vacuolization consistent with fatty change (incidences: 3, 4, 3, 7) were also observed at this dose level. The increased liver weights and liver histopathology at 159 mg/kg bw/day were considered to be adaptive and non

- adverse.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 72 of 126 Canada PMRA/US EPA: The maternal LOAEL is 323 mg/kg bw /day based on decreased body weight gains and food consumption (GD 6
- 9), incr eased liver weight , altered cytoplasmic homogeneity of centrilobular and midzonal hepatocytes, and hepatocellular vacuolization consistent with fatty change. The maternal NOAEL is 159 mg/kg bw /day.
Australia OCS: The maternal LOAEL is 159 mg/kg bw /day based on decreased body weight gains and food consumption (GD 6
- 9) , increased liver weight and altered cytoplasmic homogeneity of centrilobular and midzonal hepatocytes. The maternal NOAEL is 41 mg/kg bw /day. Developmental toxicity : No adverse externa l, visceral or skeletal malformations or variations were noted after treatment with XDE

- 279 methyl. The developmental LOAEL was not established. The developmental NOAEL is 323 mg/kg bw/day.
XDE

- 729 methyl was not present in any GD 21 dam and fetal bloo d samples. In contrast, the metabolite, XDE

- 729 acid was present at quantifiable levels (≥LLQ) in all of the blood samples from exposed animals. XDE

- 729 acid was present in fetal blood samples at concentrations ~ 65% of the dam blood levels. XDE

- 729 aci d blood concentrations were linear (dose

proportional) at all three doses in dams and fetuses

. The developmental toxicity study in the rat is classified as acceptable /guideline and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats. Nonguideline Prenatal Developmental Rangefinding Study

– Rabbit In a range

- finding developmental toxicity study (PMRA 2226502, MRID 48557839), g roups of five time

- mated female N ew Z ealand W hite rabbits were administer ed XDE

- 729 methyl (97.2% a.i., lot no. E2837

- 51, TSN031117

- 0004 ) in the diet at concentrations of 0, 521, 2083, 4167, 8333, 10417, 15625, or 24500 ppm on gestation days (GD) 7 through 28, or their date of early termination (≤GD 15), which corresponded to t ime weighted averages of 0, 21.5, 83.3, 157, 244, 371, 426, or 727 mg/kg bw /day. In

- life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed consumption. On GD 28 all surviving rabbits were euthaniz ed and examined for gross pathological alterations. Liver and kidney weights were recorded, along with the number of corpora lutea, implantations, resorptions, fetal body weights, and live/dead fetuses. Blood concentrations of XDE

- 729 methyl and the meta bolite XDE

- 729 acid were determined in samples at two time points from all does on GD 27 and at necropsy on GD 28. Based on excessive body weight loss in does treated with dietary concentrations at ≥8333 ppm (244 mg/kg bw/day), these groups were premature ly sacrificed on GD 15 since the MTD was exceeded. In the remaining groups, there were no treatment

- related effects on survival, clinical signs, kidney weights or gross pathology. Treatment

- related decreases in body weight gains (23

- 26%) and food consumpti on (≥12%) were observed in does at 83.3 and 157 mg/kg bw/day, but were not considered to be adverse due to lack of effects on mean body weight. Increased liverHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 73 of 126 weights (absolute 13

- 49%, relative 15

- 52%) were observed in females at 21.5, 83.3 and 157 mg/kg bw/day. A histological assessment of these livers was not conducted in this range

- finding study. Apart from decreased fetal weights (7

- 10%) at 83.3 and 157 mg/kg bw/day, there were no treatment

- related effects on Caesarean section parameters.
B lood toxicokinetic analysis demonstrated the presence of only the metabolite, XDE

- 729 acid , and not the parent molecule, XDE

- 729 methyl, in treated animals at concentrations above the analytical LLQ. Diurnal systemic exposure values for the metabolite XDE

- 729 a cid exhibited linear kinetics through the highest analyzed dose group (4167 ppm). Canada PMRA, Australia OCS: No LOAELs or NOAELs were established in this supplemental range
- finding study.
US EPA: A maternal NOAEL and LOAEL was identified for comparison to the definitive study. The maternal LOAEL is 244 mg/kg bw/day, based on decreased body weight, weight gain and food consumption. The maternal NOAEL is 157 mg/kg bw/day. The developmental LOAEL /NOA EL was not determined as fetal examination was not performed
. This study is classified as acceptable/non

- guideline as a range

- finding developmental toxicity study in rabbits. It was conducted to assist in dose selection for the definitive developmental s tudy and was not conducted to support a guideline requirement.
870.3700b Prenatal Developmental Toxicity Study
- Rabbit In an oral developmental toxicity study (PMRA 2226501, MRID 48557840), g roups of 24 time

mated female N ew Z ealand W hite rabbits were administered diets containing 0, 122, 391, or 1539 ppm XDE

- 729 methyl (97.2% a.i., lot no. E2837

- 51, TSN031117

- 0004) on gestation days (GD) 7 through 28, which corresponded to time

- weighted average doses of 0, 5.8, 18.5 or 71.6 mg/kg bw /day, respectively. In

- life maternal study parameters included clinical observations, body weight, body weight gain, and feed consumption. On GD 28, all rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were r ecorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. Microscopic examination of the liver was conducted for all dose groups. All fetuses were weighed, sexed, and examined for external and visceral al terations. The heads were also examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses. Blood concentrations of XDE

- 729 methyl and the m etabolite XDE

- 729 acid were analyzed in samples collected from four does/group and their respective litters at necropsy.
There were no treatment

- related effects on survival, clinical signs, body weights, food consumption or gross pathology in maternal ani mals.
In does , treatment

- related increased liver weights (absolute 9

- 27%, relative 13

- 29%) , along with increased incidences of hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of periportal hepatocytes (incidences: 1, 1 , 14, 19) , increased number of mitoticHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 74 of 126 figures in hepatocytes (incidences: 0, 0, 3, 7) and altered cytoplasmic homogeneity of centrilobular/midzonal hepatocytes (cytoplasmic glycogen) (incidences: 1, 0, 7, 12) were observed at 18.5 and 71.6 mg/kg bw /day. A t 18.5 mg/kg bw/day, these changes were considered to be adaptive liver effects and non

- adverse by Canada PMRA and US EPA, but were considered to be treatment

- related by Australia OCS. In addition, the high

- dose females had increased incidences of panlobul ar vacuolization of hepatocytes consistent with fatty change in the liver in addition to these findings . Dams in the high

- dose group had decreased gravid uterine weights at necropsy.
Canada PMRA: The maternal LOAEL is 71.6 mg/kg bw /day, based on increased liver weight, increased incidences of hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of periportal hepatocytes, increased number of mitotic figures in hepatocytes , increases in altered cytoplasmic homogeneity of centrilobular/midzonal hepatocytes (cytoplasmic glycogen) , increased panlobular vacuolization of hepatocytes (consistent with fatty change), and decreased gravid uterine weights . The maternal NOAEL is 18.5 mg/kg bw /day. Australia OCS, US EPA: The mate rnal LOAEL is 18.5 mg/kg bw /day, based on increased liver weight, increased incidences of hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of periportal hepatocytes, increased number of mitotic figures in hepatocytes and increases in altered cytoplasmic homogeneity of centrilobular/midzonal hepatocytes (cytoplasmic glycogen). The maternal NOAEL is 5.8 mg/kg bw /day.
No treatment

- related external, craniofacial or visceral malformations or variations were observed. Treatment

- related i ncreased incidences of delayed ossification of the pubis (a developmental variation ; fetal (litter) incidences: 0(0), 1(1), 6(3), 11(8) ) were observed at 71.6 mg/kg bw /day. Fetuses in the high

- dose group had treatment

- related decreased body weights (7

- 11%) upon Caesarean section but no other treatment

- related effects on pregnancy rates, litter size, numbers of corpora lutea or implantations, percent pre

- implantation loss or fetal sex ratios in any treated group were noted. Increases in resorptions/post

- implantation loss (1.2, 2.5, 6.3, 5.1%) were observed at 18.5 and 71.6 mg/kg bw/day; this finding was considered to be treatment

- related by the Canada PMRA.
Canada PMRA: The developmental LOAEL is 18.5 mg/kg bw /day, based on increases in resorptions/post
- implantation loss. The developmental NOAEL is 5.8 mg/kg bw /day. US EPA, Australia OCS: The developmental LOAEL is 71.6 mg/kg bw /day, based on decreased fetal body weights and delayed ossification of the pubis . The developmental NOAEL is 18 .5 mg/kg bw /day. Mean daily XDE

- 729 methyl intake during the period of TK data collection (GD 27

- 28) was 6, 17, and 73 mg/kg bw /day for rabbits in the 122, 391, and 1539 ppm groups, respectively. XDE

- 729 methyl was not detected in terminal blood samples of treated does or their litters. Doe blood from XDE

- 729 methyl

- treated groups contained the metabolite, XDE

- 729 acid , at levels above the LLQ. XDE

- 729 acid was also present in blood from GD 28 fetuses at levels above the LLQ in the 391 and 1539 ppm gro ups, but not in the 122 ppm group. The blood concentrationsHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 75 of 126 were dose

- proportional across all three XDE

- 729 methyl concentrations in does as determined using the least squares regression statistical test. XDE

- 729 acid fetal blood concentrations were inter preted to be less than dose

- proportional (sublinear) in the 1539 ppm group. While not statistically identified, the blood concentrations were similar to the 381 ppm group despite a 3.4

- fold higher applied dose. Fetal XDE

- 729 acid blood concentrations in the 391 and 1539 ppm groups were on average, 27 and 6% of dam blood concentrations, respectively. Thus, XDE

- 729 acid exhibited kinetic linearity in GD 28 does, in all three treated groups, but kinetic nonlinearity in GD 28 fetuses at 1539 ppm. This study is classified as acceptable /guideline and satisfies the guideline requirement for a Prenatal Developmental Study

– Rabbits; OPPTS 870.3700; OECD 414.
A.4.3 Reproductive Toxicity 870.3800 Reproduction and Fertility Effects
- Rat Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid
A.4.4 Chronic Toxicity 870.4100a (870.4300) Chronic Toxicity

– Rat Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid 870.4100b Chronic Toxicity

– Dog Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid
A.4.5 Carcinogenicity 870.4200a Carcinogenicity Study

– rat Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid
870.4200b Carcinogenicity (feeding)

– Mouse Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid
A.4.6 Mutagenicity
Gene Mutation 870.5100, Bacterial reverse mutation test, preincubation assay MRID 48557846 Acceptable/guideline 0, 50, 10, 500, 1 5 00, or 5000 μg/plate +/

- S9 Negative in Salmonella typhimurium TA 1535, TA1537, TA98, and TA100 and Esc h erichia coli Wp2 uvrA up to the limit concentration with evidence of compound insolubility but no cytotoxicity

.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 76 of 126 870.5100, Bacterial rev erse mutation test, plate

- incorporation assay MRID 48557845 Acceptable/guideline 0, 50, 10, 500, 1 5 00, or 5000 μg/plate +/

- S9 Negative in Salmonella typhimurium strains TA1537 , TA98, and TA100 and Esc h erichia coli Wp2 uvrA up to the limit concentration wit h no evidence of compound insolubility or cytotoxicity

.
870.5300, in vitro mammalian cell forward gene mutation, Chinese hamster ovary (CHO) cells, HGPRT locus MRID 48557851 Acceptable/guideline 0, 15.625, 31.25, 62.5, 125, 250 or 500 μg/m L

- S9 activation and 0, 18.75, 37.5, 75, 150, 300 or 500 μg/m L +S9 activation Negative for the induction of mutant colonies over background up to precipitating concentrations

870.53 00, in vitro mammalian cell forward gene muta tion, SD rat peripheral lymphocytes MRID 48557849 Acceptable/guideline 0, 3.13, 6.25, 12.5, 25 or 50 μg/mL+/

- S9 activation Negative for the induction of mutant colonies over background up to precipitating concentrations.

Cytogenetics
870.5375, In vitro mammalian cell chromosomal aberration assay , human peripheral lymphocytes MRID 48557854 Acceptable/guideline 0, 15.624, 31.25, 62.5, 125, 250 or 500 μg/mL +/

- S9 activation Negative for induction of chromosomal aberrations over background up to cytotoxic and precipitating concentrations.

870.5375, In vitro mammalian cell chromosomal aberration assay , primary lymphocyte cultures (Sprague Dawley rats) MIRD 48557853 Acceptable/guideline 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μg/mL

- S9 activation and 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μg/mL + S9 activation Negative for induction of chromosomal aberrations over background up to precipitating concentrations.

A.4.7 Neurotoxicity 870.6100 Delayed Neurotoxicity Study
- Hen Study not required for halauxifen

- methyl. 870.6200 Acute Neurotoxicity Screening Battery Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid
870.6200 Subchronic Neurotoxicit y Screening BatteryHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 77 of 126 Study not conducted for halauxifen

- methyl. See Executive Summaries for XDE

- 729 Acid 870.6300 Developmental Neurotoxicity Study Study not required for halauxifen

- methyl.
A.4.8 Metabolism
870.7485 Metabolism
- Rat
See Executive Summaries for XDE

- 729 acid
870.7600 Dermal Absorption
- Rat

In an in vivo dermal absorption study (MRID 48557911; PMRA No. 2226192), four male Wistar rats/dose group were dermally exposed for 10 hours to [ 14 C]

- XDE

- 729 methyl (radiochemical purity 98.5%; nonlabelled purity 98.9%, batch XW7

- 38246

- 49) formulated as GF

- 2573 (an emulsifiable concentrate), at three concentrations: 7.5 g acid equivalents (a.e.) L
- 1 (concentrate),
ca 0.15 g a.e.L - 1 (field dilution 1) and ca 0.025 g a.e.L - 1 (field dilution 2). Test material in 100 μL was applied to an area of shaved skin of 10 cm 2 to give dermal doses of 75, 1.5 and 0.25 μg/cm 2 , respectively. Following exposure the test material was rinsed off and skin was tape

stripped 20x to remove the stratum corneum. At ea ch concentration, groups were designated for termination at 24, 48, 72, 144 (concentrate only) and 192 (field dilutions) hours post

- dosing. Additionally, blood samples were collected at 4 and 10 hours post

- dosing to evaluate levels of radioactivity, and f

urther analysis was performed to determine whether XDE

- 729 methyl or its acid metabolite were present.

US EPA: Calculation of dermal absorption excluded radioactivity in the first two skin tape strips. In vivo percent dermal absorption (absorbed plus absorbable dose, excluding the outermost two layers of skin) at the end of 144

- 192 hours was 13, 15 and 24% of the applied dose (highest to lowest concentration tested, respectively). Based on the results of these studies, a dermal absorption factor (DAF) of 24% (192 hr post

- exposure) from the lowest concentration field dilution was selected as the most appropriate value for risk assessment. The 10 hr exposure duration is considered relevant for occupational exposu re and the selected value represents the maximum absorption observed in the rat in vivo absorption study.
In vitro percent dermal absorption (MRID 48557910) in the rat skin at the end of 24 hr was 4.9, 41.1 and 42.7%. The ratio of in vitro to in vivo d ermal absorption in the rat was 0.5, 3.0 and 2.2. Absorption in human skin after 24 hr was 0.8, 14.9 and 26.5%. The ratio of dermal absorption in human to rat skin was 0.16, 0.36 and 62. Canada PMRA and Australia OCS: The first two tape strips were inc luded in determination of the potentially absorbable dose for all test groups . Based on this calculation, in vivo percent dermal absorption at the end of 144

- 192 hours was 14, 18 and 30% (absorbed plus absorbable dose) of the applied dose (all values are given from highest to lowest concentration tested, respectively). A dermal absorption factor (DAF) of 30% (192 hr post

- exposure) from the lowestHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 78 of 126 concentration field dilution in the rat in vivo absorption study was selected as the most appropriate value fo r risk assessment. In vitro percent dermal absorption (MRID 48557910) in the rat skin at the end of 24 hr was 5.0, 42.5 and 45.9% of the applied dose (highest to lowest concentration, respectively). In comparison, at the end of 24 hr, in vivo dermal exp osure in the rat was 9.9, 15.7 and 23.3%. The ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 2.7 and 2.0. Absorption in human skin after 24 hr was 0.9, 15.5 and 27.9%. The ratio of dermal absorption in human to ra t skin was 0.18, 0.3 6 and 0.61. Analysis of blood samples indicated that systemic exposure following dermal dosing is to XDE

729, the acid metabolite. No parent (XDE

- 729 methyl) was identified. This study is considered acceptable/guideline and satisfies the requirement for an in vivo dermal absorption study in the rat (OPPTS 870.7600; OECD 427). Nonguideline In Vitro Dermal Absorption

– Rat and Human In a comparative in vitro dermal absorption study (MRID 48557910, PMRA No. 2226190), the in vitro percutaneous absorption of [ 14 C]

- XDE

- 729 methyl (radiolabeled purity 98.5%, batch no. XS9

- 100040

- 28; non

- radiolabelled purity 98.9%, batch XW7

- 38246

- 49), formulated as GF

- 2573 (an emulsifiable concentrate formulation) through split

- skin human and rat skin membranes was evaluated using flow

- through diffusion cells. The test substance was tested at three target concentrations (expressed as gram acid equivalents (a.e.) per liter): 7.5 g a.e.L - 1 (concentrate) and ca 0.15 g a.e. L - 1 (field dilution 1) and ca 0.025 g a.e.L - 1 ( field dilution 2). The concentrate represents the maximal concentration possible when handling the undiluted formulation, while the field dilutions reflect the concentrations recommended for use in the field. Skin samples were exposed to the test material for 10 hours, rinsed and then absorption was continued for an additional 14 hours. US EPA: The radioactivity in the first two skin tape strips was excluded from the determination of potentially absorbable dose for both the rat and human skin samples (a s presented in the study report). Based on this calculation, the in vitro percent dermal absorption in the rat skin at the end of 24 hr was 5.0, 41.1 and 42.7% of the applied dose (highest to lowest concentration, respectively). In comparison, at the end of 24 hr, in vivo dermal absorption in the rat was 9.5, 14 and 20%. The ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 3.0 and 2.2 (based on the results of MRID 48557911). Absorption in human skin after 24 hr was 0.9, 15 and 26% (hig hest to lowest concentrations, respectively). The ratio of dermal absorption in human to rat skin was 0.16, 0.36 and 0.62. Canada PMRA and Australia OCS: The first two skin tape strips were included in determination of the potentially absorbable dose. Based on this calculation, in vitro percent dermal absorption in the rat skin at the endo of 24 hr was 5.0, 42.5 and 45.9% of the applied dose (highest to lowest concentration, respectively). In comparison, at the end of 24 hr, in vivo dermal absorption i n the rat was 9.9, 15.7 and 23.3%. The ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 2.7 and 2.0. Absorption in human skin after 24 hr was 0.9, 15.5 and 27.9%. The ratio of dermal absorption in human to rat skin was 0.18, 0.36 and 0 .61. This study is classified as acceptable/nonguideline. It was performed to provide information on comparative rat and human dermal absorption and not to satisfy a guideline requirement.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 79 of 126 A.4.9 Immunotoxicity
870.7800 Immunotoxicity In an immunotoxicity study (MRID 48557864, PMRA 22265141), XDE

- 729 methyl (97.2% a.i., lot no. Lot # E2837

- 51, TSN031117

- 0004 ) was administered to ten female , F344/DuCrl rats /dose in diet at dose levels of 0, 127, 643 or 6323 ppm (0, 10 .6, 5 3.6 , or 50 5 mg/ kg/day , respectively) for 29 days. An additional group of ten female F344/DuCrl rats were included to serve as immunosuppressive positive controls (IPC) and received intraperitoneal (i.p.) doses of 20 mg/kg cyclophosphamide (CP) on study days 24

- 28. The immunotoxic potential of XDE

729 methyl was assessed through the evaluation of the primary antibody response to sheep red blood cells (SRBC) using an enzyme

- linked immunosorbant assay (ELISA) approach that measured the concentration of serum anti

- SRBC IgM

. There were no adverse effects on clinical observations, body weight, body weight gain, food consumption, hematology or gross necropsy observations. Animals given 50 5 mg/kg bw / day had statistically significantly lower mean hemoglobin concentrations, hem atocrit levels, and elevated numbers of reticulocytes when compared to those of the controls ; however, these parameters were within the historical control ranges
. Decreased absolute thymus weight and increased liver weights were observed at 505 mg/kg bw/d ay. This observed response is consistent with previously conducted 28

- and 90

- day toxicity studies which demonstrated both decreased thymic weights and the histopathological correlate of very slight lymphoid atrophy of the thymus at doses of 728 mg/kg bw /da y and 500 mg/kg bw /day, respectively. Under conditions of this study, the systemic NOAEL is 5 3.6 mg/kg bw /day, the LOAEL was 50 5 mg/kg bw /day based on increased liver weights and de creased thymus weights.
The anti

- SRBC antibody response s were within the assay range and analysis of the data did not reveal a statistically significant difference between the three doses of XDE

- 729 methyl and the vehicle control. A high inter

- individual variability was noted in all the treatment groups as well as in the contro l group. Evaluation of individual animal data of this study did not show any trend or distribution that would demonstrate significant suppression of anti

- SRBC antibody response. The positive control cyclophosphamide produced a statistically significant and greater than 99% reduction in the anti

- SRBC IgM response. The Natural Killer (NK) cell activity was not evaluated in this study. Decreased thymus weights and histopathological findings were observed in a few studies. The effects in these studies were int erpreted to be secondary to stress as the responses occurred in the presence of decreases in body weight, body weight gains and significant hepatic effects. The overall weight of evidence suggests that this chemical does not directly target the immune syst em. Under the US EPA HED guidance, a NK cell activity assay is not required at this time. The immunotoxicity NOAEL is 505 mg/kg bw/day; the LOAEL was not established. This study is classified as acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study in the rat or mouse (OPPTS 870.7800).Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 80 of 126 A.4.9 Special/Other Studies (All Nonguideline) Seven Day Dietary Study in the Rat This study (PMRA 2226494, MRID 48557879 ) was conducted to characterize the mode of action for the XDE

- 729 methyl

- induced liver and thyroid effects in the rat, and to compare the toxicokinetic profiles in rats administered XDE

- 729 methyl (97.2%, lot # E2837

- 51) versus XDE

- 729 acid (95.3%, lot # E2837

- 52) . Four male F344/DuCrl rats per gr oup were given test diets formulated to supply 0, 782 mg XDE

- 729 methyl/kg body weight/day, or 750 mg XDE

- 729 acid /kg body weight/day in the diet for seven days. Actual time

- weighted average doses were 0, 766 (XDE

- 729 methyl), or 672 mg/kg bw /day (XDE

- 729 acid ). Parameters evaluated were daily cage
- side observations, body weights, f oo d consumption, organ weights and histopatholog ical examination of the liver and thyroid, biomarker analysis of liver, blood , and urine , and targeted gene expression.
Concent rations of XDE

- 729 acid in the liver or blood from animals given XDE

- 729 acid were greater than animals given XDE

- 729 methyl at the corresponding acid equivalent dose. However, following administration of XDE

- 729 methyl most of the dose was converted to XDE

729 acid with negligible amounts of XDE

- 729 methyl recovered in the blood at 30 minutes post

dosing, and 24 hour urine (<0.03%), while 24% of the administered XDE

- 729 methyl dose was excreted in the urine as XDE

- 729 acid. The highest recovery of XDE

- 72 9 methyl was in the liver. Concentrations of four identified metabolites were lower in blood, liver tissue and urine after administration of XDE

- 729 acid versus XDE

- 729 methyl. There was evidence of higher concentrations of O

- demethyl metabolites in the l iver and urine in XDE

- 729 methyl

- treated males compared to those treated with XDE

- 729 acid. These metabolites consisted of an acyl glucuronide conjugate of XDE

- 729 acid , O

- demethyl XDE

- 729 acid , a sulfate conjugate of O

demethyl XDE

- 729 acid , and a glucuro nide conjugate of O

- demethyl XDE

- 729 acid . An additional metabolite was identified following XDE

- 729 acid administration as a glucuronide conjugate of O

- demethyl XDE

- 729 methyl. T he data suggest that induction of O

- demethylation after XDE

- 729 methyl treat ment is greater than the levels seen after XDE

- 729 acid treatment.
After XDE

- 729 acid treatment in males , there were no treatment

- related effects on thyroid and liver weights or histopathology at 672 mg/kg bw /day in males. Minor t reatment

- related decrea ses in body weight gains and food consumption were observed.
After XDE

- 729 methyl treatment in males at 766 mg/kg bw/day, t here were no treatment

- related effects on body weights, body weight gains, or f oo d consumption in males. Males had treatment

relate d increases in liver weights and very slight panlobular hypertrophy of hepatocytes and very slight increased numbers of mitotic figures in hepatocytes . In addition, these animals had treatment

- related increases in thyroid weights along with very slight hyp ertrophy of thyroid gland follicular cells and very slight increased numbers of mitotic figures in follicular cells

.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 81 of 126 T he early events in the proposed mode of action framework for XDE

- 729 methyl

- induced liver toxicity have been further characterized . After administration of 766 mg/kg bw /day XDE

- 729 methyl, the test material was present in the liver (3.99±1.04 μg/mL) and at very low concentrations in the blood (0.04±0.01 μg/mL)

. As discussed above , m ost of the administered XDE

- 729 methyl was converted to XD E

- 729 acid and the data suggest greater induction of O

demethylation after XDE

- 729 methyl treatment

. Further, all five metabolites were detected in the liver tissue and urine after dosing with XDE

- 729 methyl , with the levels of metabolites in the liver ran ging from 0.34

- 4.91 μg/mL. Gene expression profiling did reveal apparent changes (>2.0

- fold compared to control) in Cyp2b1 , Cyp2b2 , Cyp3a23 , Cyp4a22 , Ugt1a1 , Ugt1a6 or Ugt2b1 mRNA expression in males treated with 672 mg/kg bw/day XDE

- 729 acid when compared to controls. Conversely , there was a minor (4.2

- fold) increase in Cyp1a1 mRNA expression observed in XDE

- 729 methyl

- treated animals at 766 mg/kg bw/day; this finding suggest s that ligand

- induced activation of the AhR pathway occurs in XDE

- 729 methyl

- treat ed males but not in males treated with XDE

- 729 acid . The hepatic gene expression data for Cyp2b1 and Cyp2b2 also revealed a slight induction for animals administered XDE

- 729 methyl but not XDE

- 729 acid ; however, the magnitude of induction is not consisten t with a toxicologically significant activation of the CAR pathway. Cyp3a23 or Cyp4a22 transcripts were not induced in either XDE

- 729 methyl

- or XDE

- 729 acid

- treated rats indicating that the PXR and PPAR

-  signaling pathways were not activated and are likely not involved in the observed hepatic responses. Similarly, no gene expression changes were noted for either XDE

- 729 methyl or XDE

- 729 acid for the phase II glucuronidation

- associated Ugt1a1 or Ugt2b1 t ranscripts in livers of rats fed XDE

- 729 methyl . However, the phase II glucuronidation

- associated Ugt1a6 was induced in XDE

- 729 methyl treated rats but not XDE

- 729 acid treated rats and was considered possibly mediated through the AhR signaling pathway.
XDE

- 729 methyl treatment resulted in effects of liver and thyroid weights and histopathological findings that were not observed with XDE

- 729. The observed thyroid effects were considered by the study authors to be likely secondary to treatment

- related liv er alterations from induction of the AhR signaling pathway, with induction of Ugt1a6 resulting in increased hepatic clearance of T3 and T4 thyroid hormones and disruption of thyroid homeostasis

. This is supported by the m olecular analysis , which indicated hepatic activation of the AhR signaling pathway and the presence of XDE

- 729 methyl and associated metabolites in the liver but not in blood 30 minutes post

- dosing or 24 hour urine

. In contrast, the males treated with XDE

- 729 acid did not have treatment

- rel ated hepatic or thyroid effects or correlative changes in gene expression, strongly suggesting that XDE

- 729 methyl is directly associated with these hepatic and secondary thyroid effects following induction of the AhR signaling pathway

. This short

- term me chanistic study in rats is acceptable/nonguideline as a supplemental study and provides additional information relevant to characterization of the MOA of liver toxicity of halauxifen

- methyl. Seven Day Dietary Toxicity Study in the Mouse This study (PMRA 2226491, MRID 48557872) was conducted to characterize the toxicological effects of XDE

- 729 methyl (97.2%, lot # E2837

- 51 ) in the mouse , to further examine the mode of action for XDE

- 729 methyl

- induced liver effects and to examine the toxicokinetic pro file i nHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 82 of 126 mice after XDE

- 729 methyl treatment. Groups of five male and five female Crl:CD1(ICR) mice were given test diets formulated to supply 0, 261, or 782 milligrams XDE

- 729 methyl per kilogram body weight per day (mg/kg bw /day) for 7 days , which corresp onded to time

- weighted average doses of 0, 250, or 778 mg/kg bw /day for males and 0, 276, or 863 mg/kg bw /day for females, respectively.
There were no treatment

- related effects in cage

- side observations, body weights, body weight gains, food consumption , clinical chemistry parameters, kidney weights or gross observations. After XDE

- 729 methyl treatment, males and females at 778/863 mg/kg bw /day M/F had higher liver weights than controls . T he changes were considered to be treatment

- related but non
advers e (adaptive) based on the lack of histopathological correlate and effects on clinical chemistry parameters

. XDE

- 729 methyl was mostly converted to XDE

- 729 acid, which was present at quantifiable levels in all of the blood samples from treated animals. XD E

- 729 methyl was present above the lower limit of quantitation (LLQ) in ~50% of the blood samples from treated animals (taken at steady state) , but at only ≤ 1% of levels of the primary metabolite XDE

- 729 acid

. Concentrations of XDE

- 729 methyl were consid erably higher in liver than in blood , while those of the metabolite XDE

- 729 in the liver were similar to those in blood. XDE

- 729 methyl was detected in the urine only from animals in the high

- dose groups at negligible levels , while XDE

729 acid was detect ed in all urine samples at levels markedly higher than XDE

- 729 methyl. XDE

- 729 methyl quantified as XDE

- 729 acid appeared to exhibit linear kinetics in both sexes in blood, liver, and urine. The only exception to this was in blood samples from males, tak en at 6 AM on day 7. Concentrations of XDE

- 729 acid appeared to be less than dose

- proportional (sublinear) at the high dose in male mouse blood on day 7 (7 78 mg/kg bw /day). Levels of both XDE

- 729 methyl and XDE

- 729 acid were generally lower in female mic e compared to males in blood (6 am), liver and urine, despite having consumed slightly larger doses of XDE

- 729 methyl; the only exceptions were the terminal blood concentrations, where slightly higher XDE

729 methyl and XDE

- 729 acid levels were seen in fem ales. These data suggest female mice may eliminate XDE

- 729 methyl and its metabolites more efficiently compared to male mice ; however, in the absence of more robust toxicokinetic analyses, definitive conclusions on sex differences in toxicokinetic paramete rs in mice following administration of XDE

- 729 methyl cannot be made

. Levels of the major metabolite, XDE

- 729 acid , in the urine of male mice were ~76% of those observed in the urine of male rats in a previous study in male rats after 7

- day dietary treatment with XDE

- 729 methyl ( PMRA 2226494 , MRID 48557879 )

. However, o verall, the kinetics of XDE

- 729 methyl in male mice appears to be very similar to the kinetics of XDE

- 729 methyl observed in male rats. The increased hepatic Cyp1a1 levels in XDE

- 729 methyl

- treated mice and correlating treatment

- related increase in liver weights (non

- adverse) suggest that the ligand

- induced activation of the aryl hydrocarbon receptor ( AhR ) pathway seen in rats also occurs in mice. The hepatic gene e xpression data for Cyp2b10 revealed a slight induction for animals administered XDE

- 729 methyl; however, the magnitude of induction is not consistent with a toxicologically significant activation of the CAR pathway. Cyp3a11 or Cyp4a22 transcripts were not inducedHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 83 of 126 in either XDE

- 729 methyl mice indicating that the PXR and PPAR

- α signaling pathways were not activated and are likely not involved in the observed hepatic responses. In mice, XDE

- 729 methyl only caused a marginal adaptive increase in liver weigh ts in both sexes and there was no evidence of liver histopathology. This is contrasted to the study in rats where a much larger increase in liver weights were observed in addition to increased thyroid weights with correlative hepatic and thyroid histopatho logical effects observed after treatment with XDE

- 729 methyl but not XDE

- 729 acid . In rats, very slight panlobular hepatocellular hypertrophy and increased number of hepatocytes in mitosis were present in all animals and the magnitude of Cyp1a1 induction w as markedly higher than in mice. These data suggest that rats are more sensitive to XDE

- 729 methyl

- induced hepatic responses than mice.
This short

- term mechanistic study in mice is acceptable/nonguideline as a supplemental study investigating the mode o f action of halauxifen

- methyl liver toxicity. Twenty Eight Day Dietary Toxicity Study in the Rat The purpose of this short

- term dietary study ( PMRA 2226489. MRID 48557873 ) was to characterize the mode of action for XDE

- 729 methyl (97.2%, lot # E2837

- 51) induced liver effects in the rat. Groups of five male F344/DuCrl rats were exposed to 0, 3 .1 , 10 .4 , 5 4.8 or 2 7 1 mg XDE

- 729 methyl/kg body weight /day (mg/ kg bw/day) in the diet for 2 8 days. R ecovery groups were selected from the control and low dose group (0 or 10.3 mg /kg bw /day ) (4 day recovery group; n =3/dose level) and control and two highest dose groups ( 0, 5 3.7 or 2 77 mg/kg bw/day) (28

- day recovery group; n =5/dose level) were switched to control diet fo llowing a 28 day exposure before switching to cont rol diet to investigate hepatic recovery after removal of treatment.
There were no treatment

- related effects on survival, clinical signs, body weights, food consumption or kidney weights.
Treatment

- related increases in absolute and relative liver weights, hypertrophy of centrilobular/midzonal hepatocytes with altered tinctorial properties (increased cytoplasmic eosinophilia), numbers of mitotic figures (hepatocytes in mitosis) and individual hepatocellular vacuolization (consistent with fatty chang e) were observed at 54.8 and 271 mg/kg bw /day relative to controls. At 271 mg/kg bw /day, a slight increase in BrdU labeling index in the centrilobular and midzonal regions was observed after 28 days of exposure, which was considered treatment

- related when considering the related liver effects occurring at th is dose and time

- point. Examination of the gene expression responses for Cyp1a1 and Cyp1a2 revealed a dose

responsive induction profile correlating with the effects on liver weights and the appearance of histopathological apical effects in the liver consistent with ligand

- induced activation of the AhR signaling pathway at 54.8 and 271 mg/kg bw /day, respectively. Transcript levels for Cyp1 a1 demonstrated the highest induc tion of 683.8

- , and 12,180.0

- fold at doses of 5 4.8 and 2 71 mg/kg bw /day, respectively

. Similarly, the response and magnitude of the response for Ugt1a6 at 271 mg/kg bw /day is consistent with induction of this gene in other repeat dose studies (7

- day andHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 84 of 126 90

- day studies in rats) and correlat ing alterations in thyroid weights and histopathology findings indicative of activation of the AhR signaling pathway and the hepatic induction for this gene

. Consistent with hepatic inducti on of the AhR signaling pathway by XDE

- 729 methyl, not quantifiable or low hepatic concentrations of XDE

- 729 methyl were observed at 3.2 and 10 .6 mg/kg bw /day. I ncreasing hepatic concentrations of XDE

- 729 methyl were recovered in the liver at 5 4.8 and 2 71 mg/kg bw/day. Quantification of the analytes in the liver recovered six metabolites. XDE

- 729 acid was by far the most abundant analyte in all samples, followed by the glucuronide conjugate of XDE

- 729 methyl

. Previous studies with XDE

- 729 methyl have demon strated an induction of metabolizing enzymes , including phase II Ugt metabolizing enzymes with concurrent increases in liver weights with increasing dose. Consistent with this induction in the current study XDE

- 729 methyl administration result ed in increa s ed levels of conjugated metabolites of XDE

- 729 methy l in the liver when compared to administration of XDE

- 729 acid , which are rapidly eliminated in the urine. Further, the amount of XDE

- 729 acid present as the major metabolite appeared to decrease signifi cantly with increasing doses concurrent with increased recovery of conjugated metabolites with increasing dose. This is indicative of an increase in the rate of enzyme kinetics (induction of hepatic enzymes) with higher doses and is consistent with findings in the previous 90

- day study in rats ( PMRA 2226466 (amended report: PMRA 2315015). MRID 48557830 (amended MRID 49124405)). Parent XDE

- 729 methyl was not present above the lower limit of quantitation (LLQ) in any blood samples at necropsy. However , the major metabolite, XDE

- 729 acid, was present at quantifiable levels in all of the blood samples from exposed animals. Approximately 0.05% of the dose was excreted as intact parent XDE

- 729 methyl in 24 hour urine. Approximately 50, 52 and 49% of the administered dose of XDE

- 729 methyl was excreted as XDE

- 729 acid in the 24 hour urine at 10, 52 and 261 mg/kg bw/day, respectively. The total amount of XDE

- 729 excreted in urine over 24 hours was slightly lower at 3 mg/kg bw/day (44% of the administered d ose).
XDE

- 729 methyl and all six of its measured metabolites were present in liver at levels above the lower limit of quantification (LLQ) in all but the low (3.1 mg/kg/day) dose group, which did not contain quantifiable levels of XDE

- 729 methyl or the s ulfate conjugate of O

- demethyl XDE

729. The metabolites included de

- methylated XDE

- 729 methyl (XDE

- 729), which was the major metabolite, and five minor metabolites, which were demethylated and/or conjugated XDE

- 729 methyl and/or XDE

- 729. XDE

- 729 was by f ar the most abundant analyte in all samples, followed by the glucuronide conjugate of XDE

- 729 methyl. At 3.1 mg/kg/day, XDE

729, and all of the glucuronidated metabolites were present. XDE

- 729 methyl was not present above LLQ in liver at 3.1 mg/kg/day. X DE

- 729 methyl levels were approximately 10% of acid levels in liver at 10.4 and 54.8 mg/kg/day. In the 271 mg/kg/day dose group, XDE

- 729 methyl levels were 20% of XDE

- 729 acid levels and appeared to be supralinear. As observed previously, the percent of t he administered dose present as the major metabolite, XDE

- 729, in the liver at the time of necropsy, decreased significantly with increasing doses. Statistically significant sublinearity in the dose

- liver level relationship was identified in the 271 mg/kg /day dose group. Conversely, the percent of the administered dose present as conjugated metabolites appeared to increase with increasing dose, though the increase was not statistically significant. XDE

- 729 methyl was dose

- proportional across all doses in liver and urine but appeared slightly supralinear at 261 mg/kg/day in liver. Parent XDE

- 729 methyl was not present above LLQ in any blood samples at necropsy. However, the major metabolite, XDE

- 729, was present atHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 85 of 126 quantifiable levels in all of the blood samples from exposed animals. Blood levels of XDE

- 729 were significantly less than dose

- proportional (sublinear) at 271 mg/kg/day. Approximately 50% of the administered dose of XDE

- 729 methyl was excreted as XDE

- 729 in the urine. The total amount of XDE

- 729 excreted in urine over 24 hours declined with increasing doses and was statistically identified as sublinear at 271 mg/kg/day. The two recovery groups demonstrated complete reversal of c hanges in liver weight, liver histopathology, Cyp1a1 , Cyp1a2 , C yp2b1 and Ugt1a6 mRNA expression at all doses tested after a 4 or 28 day recovery period. BrdU uptake in hepatocytes appeared to be still slightly elevated compared to the baseline range of labeling index (LI) values, but lower than the LI values following 28

- days of treatment suggesting at least a partial recovery. However, based on discrepancies in the data, reversibility of the increased proliferative response at 277 mg/kg bw/day could not be adequately interpreted. There were no quantifiable liver conce ntrations of XDE

- 729 methyl or XDE

- 729 in the blood, liver or urine after the specified recovery period. The data from this study further support an AhR

- mediated mode of action for the hepatic responses observed af

ter exposure of rats to XDE

- 729 methyl. T he recovery for each of the responses highlights the transient and ligand

- mediated nature of the hepatic effects due to a lack of sustained activation of the AhR signaling pathway.
This study is considered acceptable/nonguideline as a supplemental stu dy to provide information for characterization of the mode of action for liver toxicity from halauxifen

- methyl. In Vitro Transactivation Assay and Ligand Binding Study
In an in vitro transactivation assay and ligand binding study ( PMRA 2226490, MRID 48557878), XDE

- 729 acid (95.3%, lot # RSMP 1077053) , XDE

- 729 methyl technical (97.2% , lot # RSMP 1076831, ), XDE

- 729 methyl (99.1% , lot #RSMP 1076831 ), and XDE

- 729 methyl ultrapure (99.7% a.i., lot # RSMP 1082768) were tested in mouse and human stable cell lines containing a luciferase reporter vector driven by dioxin

- responsive elements that are responsive to AhR agonists. 2

- (1 ́H

- indole

- 3 ́

- carbonyl)

- thiazole

- 4

- carboxylic acid methyl ester (ITE, 99.9%), a potent AhR agonist, was included as a positive contr ol and a reference for relative potency. XDE

- 729 acid was tested for AhR agonist activity in duplicate using Hepa 1.1 cells (mouse) and Hep G2 40/6 cells (human). In all experiments, XDE

- 729 acid failed to exhibit any agonist activity. XDE

- 729 methyl (97. 2%) was assayed in duplicate using Hepa 1.1 cells (mouse) and in Hep G2 40/6 cells (human). In Hepa 1.1 cells, XDE

- 729 methyl technical (97.2%) exhibited significant agonist activity at 10 and 50  M. The XDE

- 729 methyl (99.1%) exhibited similar agonist ac tivity to XDE

- 729 methyl (97.2%). In assays using Hep G2 40/6 cells, both XDE

- 729 methyl (97.2%) and XDE

- 729 methyl (99.1%) failed to induce significant AhR tran sactivation activity.
XDE

- 729 methyl ultrapure (99.7%) mediated induction of AhR activity in a dose

- dependent manner at 10 and 50 μM in the mouse reporter cell line. In contrast, XDE

- 729 methyl ultrapure exhibits essentially no activity in the human reporter cell line at the doses tested. The data obtained with XDE

- 729 ultrapure (99.7%) was essent ially the same as with XDE

- 729 methyl (97.2%) and XDE

- 729 methyl (99.1%). These data indicate that XDE

- 729 methyl and not anHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 86 of 126 impurity is a weak activator of the mouse AhR, but not of the human AhR. However, it must be noted that the positive control, ITE, also produced a much weaker response with the human Hep G2 40/6 reporter cell line. This makes it difficult to make comparisons of the ability of XDE

- 279 methyl to be a weak agonist of the human AhR compared to the mouse AhR. Overall, the positive control (ITE) demonstrated a dose dependent increase in AhR activity at all concentrations confirming the sensitivity and validity of the assay.
To determine if XDE

- 729 methyl ultrapure (99.7%) is an actual AhR ligand that binds to the ligand

- binding pocket, a com petition AhR ligand binding assay was performed. The results indicate that XDE

- 729 methyl ultrapure (99.7%) is an AhR ligand that weakly competes with the photoaffinity ligand for the rat Ah receptor. These results are consistent with the results obtained in the reporter assays.
This in vitro mechanistic assay in mouse and human liver cells is acceptable/nonguideline as supplemental data submitted to characterize the mode of action of liver toxicity from halauxifen

methyl. In Vitro Expression of Cyp Genes and AhR Activation in Cultured Hepatocytes from Mice, Rats and Humans The objective of this in vitro mechanistic study (PMRA 2226487, MRID 48557876) was to assess the potential of XDE

- 729 methyl (99.1%, lot # not reported ) to induce Cyp1a1 /CYP1A1 and Cyp1a2

/CYP1A2 in primary cultures of human, mouse and rat hepatocytes. Evaluation of the degree of CYP induction was performed by measuring and comparing mRNA levels between the various treatment groups by RT

- PCR (TaqMan). Freshly isolated hepatocytes fro m six separate human donors were cultured on collagen

- coated plates and incubated with control inducers, vehicle controls, or test article for varying amounts of time up to 24 h ou rs . The same was done for mouse and rat hepatocytes using 3 separate pools of 3 mice and 3 rats each. Samples at 6 and 24 hours were also taken to assay for Lactate Dehydrogenase (LDH) release as a marker of toxicity using the Promega CytoTox

- ONETM Homogeneous Membrane Integrity System. Gene expression of Cyp1a1 /CYP1A1 and Cyp1a2 / CY P1A2 were analyzed using RNA prepared at the specified time points. Control induction experiments were conducted using a dose response of 3

- methylcholanthrene (3

- MC) and 1 mM phenobarbital (PB). The 3

- MC controls were quantitated for CYP1A or Cyp1a express ion levels and the PB samples were assayed for CYP2B or Cyp2b expression levels to monitor induction in the isolated hepatocytes

. A dose responsive increase in primarily Cyp1a1 /CYP1A1 was observed with the 3

- MC positive control. Cyp1a1 /CYP1A1 was more re sponsive compared to Cyp1a2 / CYP1A2 with the mouse and human, but Cyp1a2 was induced to a greater extent in the rat. For Cyp1a1 /CYP1A1 induction, mouse was the most sensitive, followed by the human where all but 1 donor had a higher CYP1A1 induction than the rat. The rat was most responsive to 3

- MC with respect to Cyp1a 2/CYP1A2 induction, the human was the next most responsive and the mouse had the least amount of Cyp1a2 induction. After treatment with another positive control, PB, there wa s induction of Cyp2b10 in the mouse, Cyp2b6 in the rat and CYP2B6 in human hepatocytes. The response in the mouse was modest and markedly lower than expected. No further studies were conducted to discern a cause for the dampened response; however, based on the robust increaseHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 87 of 126 in Cyp1a mRNA levels in the treatment groups, this deficiency is not expected to invalidate the methods used in this study. Overall, the positive control results confirmed the validity and sensitivity of the assay for Cyp1a /CYP1A and C

yp2a/CYP2A induction in mouse, rat and human hepatocytes.
XDE

- 279 methyl was also able to induce Cyp1a1 /CYP1A1 and Cyp1a2 / CYP1A2 expression in hepatocytes of all species, but at levels lower than the positive control. The r at was the most responsive to Cyp1a1 /CYP1A1 and Cyp1a2 / CYP1A2 induction compared to mouse and human hepatocytes that had limited induction of Cyp1a1 /CYP1A1 and Cyp1a2 / CYP1A2 with XDE

- 729 methyl across multiple dose levels up to 100 μM and multiple time

- points up to 24 hours compared to the positive control, 3

- MC. All species were generally more sensitive to Cyp1a1 /CYP1A1 induction compared to Cyp1a2 / CYP1A2 induction following XDE

- 729 methyl exposure. Rat hepatocytes ( most responsive to XDE

- 729 methyl) had Cyp1a1 and Cyp1a2 maximum ind uctions slightly lower in responsiveness compared to the positive control. Average induction at the 2 highest concentrations was 32

- fold and 13

- fold for Cyp1a1 and Cyp1a2 , respectively. Human hepatocytes were less responsive than mouse and rat hepatocytes to Cyp1a1 /CYP1A1 induction with an average of 4

- fold induction increase at the two highest concentrations compared to negative controls but were slightly more responsive to Cyp1a2 / CYP1A 2 induction compared to mice with an average of 1.5

- fold increase at the two highest concentrations compared to negative controls . Peak expression of the examined Cyp1a1 /CYP1A1 isoforms occurred from 8

- 12 hours. All species were generally more sensitive to Cyp1a1 /CYP1A1 induction compared to Cyp1a2 / CYP1A2 induction. No significant cytotoxic effect was observed in any species with any treatment as demonstrated by the lack of lactate dehydrogenase ( LDH ) leakage

. T his in vitro mechanistic assay in mouse, r at and human hepatocytes is acceptable /nonguideline as supplemental data. It provides additional information to characterize the mode of action for liver toxicity from halauxifen

- methyl. In Vitro Hydrolys is Rates, Liver S9, Blood a nd Synthetic Gastric Fluid of Mouse, Rat, a nd Human a nd PBP K Simulations o f Systemic Exposure i n Rats a nd Humans
In this in vitro mechanistic study (PMRA 2226488, MRID 48557874) , r ates of metabolism of XDE

- 729 methyl (97.2%, Lot# E2837

- 51) to XDE

- 729 acid (95.3%, Lot # E2837

- 52) were determined by conducting in vitro incubations of the XDE

- 729 methyl in key physiological matrices (liver S9 ce ll fraction, whole blood, and synthetic gastric fluid (SGF)) of mouse, rat, and human. These rates were then incorporated into a whole

- body, physiologically

- based pharmacokinetic (PBPK) model to predict systemic exposures to XDE

- 729 methyl and XDE

- 729 in blood and liver tissue of rats and human.
XDE

- 729 methyl was rapidly hydrolyzed to XDE

- 729 acid by liver S9 of all three species at c oncentrations up to 10 μM. Metabolism of XDE

- 729 methyl by liver S9 appeared to follow (saturable) Michaelis

- Menten kinetics; thus, K m and V max terms were derived to mathematically define the metabolism of XDE

- 729 methyl in liver tissue. The data demonstr ated relative rates of metabolism decreasing in order of (fastest to slowest) human>>rat>mouse. The rate ofHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 88 of 126 metabolism was so rapid in incubations with human liver S9 that XDE

- 729 methyl was not present at levels exceeding the limit of quantitation after 8 minutes of incubation. The mass balance data indicated that XDE

- 729 acid was the primary metabolite of XDE

- 729 methy l, and furthermore, that there was minimal downstream metabolism, by liver S9, of XDE

729 acid

. This was also observed in analyses of whole blood.
XDE

- 729 methyl was metabolized in whole blood according to 1st order kinetics, with the relative rates between species following the order of (fastest to slowest) rat>mouse>>human. The rate of metabolism of XDE

- 729 methyl in SGF was the slo west of all test media evaluated. The fastest rate of conversion occurred in SGF at pH 1.2 (representing the pH of human GI tract), with a half

- life of approximately 100 hours; again, this was much slower than the rates of metabolism determined for liver S9 and whole blood

. The in vitro

- derived rates were incorporated as parameters into a PBPK model, and the model adapted to simulate 28

- day dietary exposures of XDE

- 729 methyl to rats and humans. The predicted hydrolysis of XDE

- 729 methyl was most depen dent on the rate of metabolism in liver tissue for both species, accounting for ~60% of total hydrolysis in rat and >95% in human. The model simulations indicate that XDE

- 729 methyl and XDE

- 729 acid achieve their steady

- state concentrations soon after exp osure is begun, the levels clear rapidly from blood and liver following the cessation of exposure, and that neither XDE

- 729 methyl nor XDE

- 729 acid would be expected to accumulate in either liver or blood following exposures to XDE

- 729 methyl. In a m odel

- derived 28

- day simulation at 10 mg/kg bw/day peak concentrations (and 24 h AUCs) of XDE

- 729 methyl in liver and blood are comparable between rats and human. The peak concentrations from a simulated 28

- day exposure to XDE

- 729 methyl at 0.0001 m g/ k g bw/ d ay, a relevant exposure level based on residue data, resulted in predicted levels of XDE

- 729 methyl in humans that are 10000

- to 15000

- times lower than the limits of quantitation (LOQs: in blood ~0.01 μg XDE

- 729 methyl/g; in liver ~0.04 μg XDE

- 729 methyl/g).
This in vitro mechanistic study is considered acceptable/nonguideline as a supplemental study. It provides information used to identify a mode of action for liver toxicity of halauxifen

- methyl. Discussion of MOA and Human Relevance Framework Analysis
XDE

- 729 methyl induces rodent liver effects via a proposed aryl hydrocarbon receptor

- mediated mode of action (MOA) through the following key events (1) presystemic liver exposure to XDE

729 methyl, (2) aryl hydrocarbon receptor (AhR) activation with associ ated liver weight increase and hepatocyte hypertrophy, leading to (3) hepatocellular proliferation. These key events have been evaluated in a series of guideline toxicity and MOA studies aimed at characterizing the MOA for XDE

- 729 methyl

- mediated liver ef fects. This document (PMRA 2226486, MRID 48557875 ) represents the weight of evidence approach used to evaluate the data based upon the Bradford

- Hill criteria , followed by subsequent application in a Human Relevance Framework (HRF). Following dietary administration to rodents, XDE

- 729 methyl is rapidly hydrolyzed to the primary metabolite XDE

- 729. Although XDE

- 729 methyl is an AhR activator, XDE

- 729 has been shown through both in vitro and in vivo assays not to induce AhR activation and associatedHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 89 of 126 liver responses. Therefore, this MOA/HRF analysis is focused on XDE

- 729 methyl induced AhR

- mediated effects in the liver. Presystemic exposure of the rodent liver to XDE

- 729 methyl results in a dose responsive increase in Cyp1a1 transcript levels as a sensitive biomarker for AhR activation. In addition, corresponding increases in liver weight and associated hepatocellular hypertrophy were observed. Supporting events for an AhR

- mediated MOA for hepatocellular effects include ligand binding to the receptor , and a series of in vitro assays , which have collectively indicated that XDE

- 729 methyl is a weak AhR agonist. Presystemic liver exposure to XDE

- 729 methyl, followed by activation of rat AhR, leads to liver weight increases with hepatocel lular hypertrophy and increases in hepatocellular proliferation. Importantly, these effects (i.e., measureable molecular and apical end points) occur at doses ≥50 mg/kg bw/day XDE

- 729 methyl with a clear threshold for liver adverse effects at a no

- observe d

- adverse

- effect level (NOAEL) of 10 mg/kg bw/day, and for Cyp1a1 induction, of 3 mg/kg bw/day, based on mild induction (52

- fold) at 10 mg/kg/day in the absence of other observable liver effects. The data from these evaluations provides a high level of co nfidence that XDE

- 729 methyl induces rodent liver effects through an AhR

- mediated MOA.
C omprehensive toxicokinetic analysis in the course of the studies evaluated in this MOA/HRF determined that XDE

- 729 methyl is rapidly metabolized and excreted in the urine following dietary administration. Low levels of XDE

- 729 methyl were present in the liver following dietary administration; however, after a four or 28 day recovery period, there was no XDE

- 729 methyl present in the livers of rats.
A physiological ly

- based pharmacokinetic ( PBPK ) model for XDE

- 729 methyl exposure predict ed similar levels of XDE

- 729 methyl in the livers of humans and rats at the overall NOAEL from the toxicity studies of 10 mg/kg bw/day, as well as the conservative estimated human exp osure scenario of 0 .0001 mg/kg bw /day. Although PBPK model outputs indicate that human liver would be exposed to XDE

- 729 methyl, the concentrations modeled are well below those that would cause hepatic effects based on rodent toxicity studies (~50 mg/kg b w/day). Furthermore, there is a quantitative difference of ~10

- fold for binding affinity of the prototypical AhR ligand dioxin to human versus rodents AhR, which would suggest that animal models would likely be more sensitive to AhR

- mediated effects than humans (Connor and Aylward, 2006). In addition, the doses causing hepatic toxicity in rats are considered to be well above those that would cause hepatic effects in humans based on quantitative differences in sensitivity to AhR activation and corresponding toxicity (discussed below).
Several in vitro studies for AhR receptor activation were also completed as part of the assessment of human relevance. A conclusion from the rodent MOA data and in vitro mechanistic studies is that humans would be signifi cantly less sensitive than rat to XDE

- 729 methyl based on the following: 1 ) kinetic analysis of XDE

- 729 methyl indicated this molecule is rapidly hydrolyzed to XDE

- 729 by human liver S9 and does not bioaccumulate in rat liver tissues in which hydrolysis i s substantially slower , 2 ) in vitro transactivation and binding assays with XDE

- 729 methyl indicated that this molecule was a relatively weak ligand for rodent AhR and ha d no transactivation activity for human AhR, 3) at concentrations of XDE

- 729 methyl that elicited substantial induction of Cyp1a1 transcript levels in the rat, primary human hepatocytes had minimal induction of CYP1A1 and CYP1A2. In addition, a clear threshold for adverse liver effects exists for XDE

- 7 29 methyl in rodents with c omprehensive MOA and mechanistic dataHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 90 of 126 indicating that molecular and apical endpoints ( Cyp1a1 induction, liver enlargement with associated hypertrophy, and hepatocellular proliferation) do not occur below the dose level of 10 mg/k g bw /day XDE

- 729 methyl in 28 day to 90 day repeat dose dietary studies

. The data identify a dose (3 mg/kg bw/day) at which only slight Cyp1a1 induction is observed and which is well below the LOAEL for liver toxicity (52 mg/kg/day). This dose is conside red protective for long

- term exposure since the initial event for liver toxicity is not observed. The data from these evaluations provide a high level of confidence that XDE

- 729 methyl induces rodent liver effects through an AhR

- mediated MOA. Although XD E

- 729 methyl was able to activate the AhR, it is a weak agonist that is rapidly metabolized; therefore, XDE

- 729 methyl is not likely to result in sustained activation of the AhR signaling pathway. In addition, a ll hepatic molecular and apical end points w ere reversible after short recovery periods. Analysis of toxicity, toxicokinetic and molecular end points following XDE

- 729 methyl administration indicated a clear threshold for liver effects. In vitro transactivation studies and gene expression studies in human liver cells also demonstrated minimal activation of the human AhR with exposure to XDE

- 729 methyl, which is coupled by the fact that in vitro hydrolysis studies using human and rat liver S9 determined that hydrolysis of XDE

- 729 methyl is faster in the human compared to the rat. Although the human relevance of an AhR

- mediated MOA conservatively cannot be excluded , the mechanistic and PBPK data strongly support the presence of a threshold to the key events in rodents that would be protective of human health

. XDE
- 729 ACID
A.4.1 Subchronic Toxicity
870.3050 28
- Day Oral Toxicity
- Rat In a subchronic oral toxicity study (PMRA 2226460, MRID 48557825), XR

- 729 acid (99.0% a.i., lot no. E2350

- 93, TSN030751

- 0002) was administered to 5 Fischer 344 rats /sex/group in the diet at dose levels formulated to supply 0, 10, 50, 250 or 1000/500 m g/kg/day for at least 28 days ( time

- weighted average concentrations : 0, 10.5, 52.7, 270 or 734 mg/kg/day for males and 0, 10, 50.3, 250 or 982 mg/kg/day for females, res pectively )

. In addition to the standard toxicity parameters, a toxicokinetic analysis of the test material in blood (collected at three time points) and urine (collected overnight) was performed during the final week of the study.
There were no treatment

- related effects on survival, ophthalmoscopy, hematology, clinical chemistry or gross pathology. Males at 734 mg/kg/day had body weight loss and decreased food consumption (↓9% from controls) during the first eleven days of the study due to reduced pala tability of the diet; therefore, the targeted dose level for these males was reduced from 1000 to 500 mg/kg bw/day (targeted doses) for Days 11

- 29 with an achieved mean dose of 734 mg/kg bw/day for both targeted dose levels. After the reduction in the die tary concentration, the amount of food consumed increased to control levels and although animals began to gain weight, overall, the high

- dose male body weights were still lower than the controls throughout the remainder of the study. Females at 982 mg/kg b w/day also had lower body weights and food consumption values.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 91 of 126 One of the males that exhibited decreased body weights also appeared thin upon clinical examination. High

- dose males and females had higher urine volumes (♂ ↓84%, ♀↓41% from controls) and lo wer specific gravity (♂ ↓0.028, ♀↓0.014 from controls) than the controls. These alterations were associated with treatment

- related kidney lesions. In addition, high

- dose females had cloudy urine and crystalline phosphate in the urine sediment. Final bod y weights were lower in males and females from the high

- dose groups. This was accompanied by increased relative kidney weights (♂ ↓6% from controls). The kidneys of high

- dose males and females had treatment

- related histopathological tubular effects, whic h consisted of tubular degeneration (incidences ♂ 0, 0, 0, 0, 5, ♀ 1, 0, 1, 0, 4 in the respective dose groups), and hypertrophy (all animals affected vs. 0 in control and lower dose groups) and vacuolization of collecting duct epithelium (incidences 1♂, 4 ♀ vs. 0 controls). Splenic extramedullary hematopoiesis was also observed in 4 high

- dose males and one high

- dose female. The LOAEL is 734 mg/kg bw /day in males and 982 mg/kg bw /day in females, based on decreased body weight, body weight gains and food consumption, increased urinary volume, decreased urine specific gravity, increased relative kidney (males only) weights, renal histopathology (degeneration and hypertrophy of renal tubules and vacuolization of collecting duct epithelium) and splenic extram edullary hematopoiesis. Females also exhibited cloudy urine and phosphate crystals in urine sediment. The NOAEL is 270 mg/kg bw /day in males and 250 mg/kg bw /day in females. Absorption of the XR

- 729 acid test material from the diet appeared to be slightly saturated at the top three dose levels in both sexes. The ingested XR

- 729 acid was rapidly eliminated from plasma upon the removal of the fortified diet (fasting) and dropped below the limit of quantification after ~16 hours of fasting for all dose group s. On Day 23 (day of blood collection) the calculated intake were 11, 53, 286, 562 mg/kg bw /day for males and 10, 47, 233, 925 mg/kg bw /day for females. T he increase in the s ystemic dose (AUC 24h ) vs. administered dose demonstrated approximately linear proportionality. Total u rinary elimination of XR

- 729 acid and metabolites at 24 hours post

- dosing demonstrated a slight decrease in female rats at the highest two dose levels of 233 and 925 mg/kg bw/day compared to the two lower dose groups. In total, male rats eliminated in urine an average of 53

- 63% of what they consumed during a 24

- hour period, while elimination by females averaged 48

- 69%. Overall, these pharmaco kinetic data s how that urine is a major elimination pathway in rats following dietary ingest ion of XR

- 729 acid after 28 days of continuous exposure and indicate that at 24 hours post

- dosing decreased elimination in urine at high doses in females

. In addition to parent XR

- 729 acid, four metabolites (glucuronide conjugate of O

- demethyl XR

- 729 acid, sulfate conjugate of O

demethyl XR

- 729 acid, O

- demethyl XR

- 729 acid, and acyl

- glucuronide conjugate of parent XR

729 acid) were tentatively identified in urine. This identifie d metabolites were comparable to those observed following single dose oral gavage administration of XR

- 729 acid at 1000 mg/kg bw. This subchronic toxicity study in the rats is acceptable /non

- guideline and satisfies the guideline requirement (OECD 407) fo r a repeat

- dose oral study in rats.
870.3100 90
- Day Oral Toxicity
- RatHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 92 of 126 In a subchronic oral toxicity study (MRID 48557828, PMRA 2226467), 10 F344/DuCrl rats /sex/group were given test diets formulated to supply XDE

- 729 acid (96.5% a.i., lot no. E2978

- 05

- 01, TSN030571

- 0005) at concentrations of 0, 10, 50, 250, or 750 mg/kilogram body weight/day (mg/kg bw /day) for 9 3

- 94 days. Average intake of XDE

- 729 acid corresponded to time

- weighted average concentrations of 0, 10.5, 52.4, 262, or 782 mg/kg bw /day for males and 0, 10.1, 50.3, 252, or 758 mg/kg bw /day for females, respectively. In addition to standard guideline parameters, toxicokinetic analyses were performed on blood (sampled at three time points) and urine (collected overnight) sampled during the las t week of treatment.
There were no treatment

- related effects on survival, clinical signs, food consumption, hematology, clinical chemistry, ophthalmoscopic parameters or gross pathology.
Treat ment

- related decreases in body weight (6%) and body weight gains (9%) were observed in high

- dose males. Males at 782 mg/kg bw/day also had increased urinary volume s (♂ 162%, ♀ 89%) , decreased urine specific gravity (♂ 0.03, ♀ 0.02), and increased renal his topathological lesions including tubular dilatation and degeneration (incidences: 0, 0, 0, 0, 8). The relative testes, thyroid, kidney and brain weights in high

- dose males were higher than the concurrent control values (7

- 9% each) and were considere d to be secondary to the lower mean final body weight. Both sexes of rats at the high

- dose level exhibited renal necrosis (incidences: ♂ 0, 0, 0, 0, 3; ♀ 0, 0, 0, 0, 1) , increased mitotic figures (incidences: ♂ 0, 0, 0, 0, 4) , hypertrophy of the collecting duct e pithelial cells (incidences: ♂ 0, 0, 0, 0, 10; ♀ 0, 0, 0, 0, 5) and vacuolization of the collecting duct epithelium in the papilla of the kidney (incidences: ♂ 0, 0, 0, 0, 10; ♀ 0, 0, 0, 0, 8). The incidence and severity was increased in males compared to females. The high

- dose females exhibited amorphous crystals in urine sediment at the urinalysis. The LOAEL in males is 782 mg/kg bw /day, based on decreased body weight and body weight gains, increased urinary volume, decreased urine specific gravity and r enal histopathology (necrosis, increased mitotic figures, hypertrophy of collecting duct epithelial cells, vacuolization of collecting duct epithelium in the papilla of the kidney, tubular dilatation and degeneration). The LOAEL in females is 758 mg/kg bw /day, based on increased amorphous crystals in the urine sediment and renal histopathology (necrosis, increased mitotic figures, hypertrophy of collecting duct epithelial cells, vacuolization of collecting duct epithelium in the papilla of the kidney). The NOAEL is 262 mg/kg bw /day in males and 252 mg/kg bw /day in females. Based on the plasma concentrations of XDE

- 729 acid , the absorption and systemic bioavailability of XDE

- 729 acid were dose

- proportional and comparable between both male and female rats. The amount of XDE

- 729 acid eliminated in the urine of male rats increased proportionally with dose; however, there was sub linearity in urinary excretion of test material in females between the 2 52 and 75 8 mg/kg bw /day.
This subchronic oral toxicity study in the rats is classified as acceptable /guideline and satisfies the guideline requirement for a subchronic toxicity study (OPPTS 870.3100; OECD 408) in rats.
870.3050 28
- Day Oral Toxicity

– MouseHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 93 of 126 In a subchronic oral toxicity study, XR

- 729 acid (MRID 48557827; PMRA 2226464) (99.0% a.i., lot no. E2350

- 93, TSN030751

- 0002) was administered by diet to 5 Crl: CD

- 1 (ICR) mice /sex/ group at target concentrations of 0, 10, 50, 250 or 1000 m g/kg bw/day for 31 days (time

- weighted average concentrations: 0, 10.2, 5 4.0, 269 or 1025 mg/kg bw /day for males and 0, 9.9, 48.7, 241 or 958 mg/kg bw /day for females , respectively ). Steady

- state concentrations of the parent chemical in the blood were determined to characterize the relationship between the oral dose and interna l systemic dose

. Recovery of parent chemical in the urine was measured to characterize the relationship between the oral dose and the efficacy and dose proportionality of urinary elimination. Potential metabolites were not characterized in this study.
Th ere were no mortalities or clinical signs and no treatment

- related effects on body weight , body weight gain, food consumption , or ophthalmology

. In females there were no treatment

- related changes in hematological parameters, organ weights, and no treatment

- related gross or histopathological findings. In both sexes, the internal systemic dose in the blood plasma was proportional to the oral dose across all dose levels. Similarly, there was no overt effect of treatment dose level on the apparent fraction of t he oral dose that was eliminated via the urine. Decreased urinary elimination of XR

- 729 acid at the high dose in males was considered an indication of a slight non

- adverse effect on kidney function. There was no evidence of saturation effects in either the steady

- state systemic exposure or the daily urinary elimination of the parent chemical. In contrast to females, m ales dosed at 1000 mg/kg bw /day had non
- adverse treatment

- related decrease s in red blood cell count (8%) and increases in the mean cell volum e of red blood cells (RBC) (7%)

. The US EPA considered the RBC findings as a slight treatment

- related effect but also note d that RBC findings were not observed in the 90

- day mouse study and were not evaluated in the onco genicity study . In both males and f emales there was a slight treatment

related decrease in serum ALT levels (♂ 33%, ♀ 26%) at the highest dose tested; this finding was considered to be treatment

- related by Canada PMRA and Australia OCS. In males receiving 1000 mg/kg bw/day, decreased kidney weight (absolute 12%, relative 12%) was concordant with an increased incidence of kidney tubule degeneration. The males at this dose level also exhibited increased incidences of multifocal karyomegaly (incidence of 1 vs. 0 in controls) and focal necrosis (incidence of 2 vs. 0 in controls) in the liver.
The LOAEL is 1025 mg/kg/day in males , based on decreased alanine aminotransferase activity (Canada PMRA/Australia OCS), decreased numbers and mean volume of red blood cells (US EPA), decreased weight and an increased incidence of tubule degeneration in the kidney (Canada PMRA/Australia OCS), and increased incidences of multifocal karyomegaly and focal necrosis in the liver. The LOAEL is 958 mg/kg bw/day in females , based decreased alanine aminotransferase activity. The NOAEL is 2 69 mg/kg bw /day in males and 241 mg/kg bw /day in females. This subchronic toxicity study in the mice is classified as acceptable /guideline and satisfies the guideline requirement (OECD 407 ; OPPTS 870.3050 ) for a repeat

- dose oral study in mice

. 870.3100 90
- Day Oral Toxicity

– MouseHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 94 of 126 In a subchronic toxicity study (MRID 48557829; PMRA 2226468), t en male and ten female Crl:CD1(ICR) mice per group were given test diets formulated to supply 0, 50, 250, 500 1000 milligrams XDE

- 729 acid (96.5% a.i., lot no. E2978

- 05

- 01/TSN030751

- 0005) per kilogram body weight per day (mg/kg bw /day) for at least 90 days which corresponded to time

- weighted average doses of 0, 49.4, 247, 495, or 989 mg/kg bw /day for males and 0, 50.2, 258, 512, or 1008 mg/kg bw /day for females, respectively. Parameters evaluated were daily cage

- side observations, weekly detailed clinical observations, ophthalmic examinations, body weights, f oo d consumption, blood and urine collectio n for toxicokinetics, hematology, clinical chemistry, organ weights, and gross and histopathologic al examinations.
There were no treatment

- related effects on survival, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights or gross pathology. In high

- dose males, treatment

- related urinary bladder toxicity [ acute inflammation (incidences: 0, 0, 0, 0, 2) , submucosal edema (incidences: 0, 0, 0, 0, 2) , hyperplasia of transitional epithelium (incidences: 0, 0, 0, 0, 1) , mu ltiple foci of ulceration (incidences: 0, 0, 0, 0, 1) , crystals (incidences: 0, 0, 0, 0, 1)] was observed and in one male, was associated with seminal vesicle acute unilateral inflammation. No urinary bladder effects were observed in females at any dose le vel. An increase in the proportion of neutrophils in the blood (↑31%) w as observed in 989 mg/kg bw/day males and w as considered secondary to urinary bladder inflammation

. The LOAEL is 989 mg/kg bw /day, based on urinary bladder histopathology and a second ary associated increase in neutrophils in males . The LOAEL in females was not establi shed, as there were no adverse effects noted up to the limit dose. The male NOAEL is 495 mg/kg bw /day and the female NOAEL is 100 8 mg/kg bw /day.
Linear kinetics were obse rved in the plasma and urine after XDE

- 729 acid exposure, although high variability was noted in the female 512 mg/kg bw/day urine group . On average, 3 2

- 47% of the XDE

- 729 acid dose was excreted in the 24 h urine . There were n o apparent sex differences in the toxicokinetic parameters examined. This subchronic toxicity study in the mice is classified as acceptable /guideline and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in mice. Nonguideline Oral Rangefinding

– Dog In a palatability an d range

- finding toxicity study (MRID 48557871, PMRA 2226465) , XR

- 729 acid (99.0% a.i. , lot no.E2350

- 93, TSN030751

- 0002 ) was administered to Beagle dogs in diet at dose levels of 0 or 7500 ppm for 3

- day consecutive ex posure periods during the palatability phase. Initial dose levels were subsequently escalated or de

- escalated to assess palatability and determine dose levels for the 28

- day phase. At 30000 ppm during the palatability phase, the females did not eat suffici ent quantities, therefore, the females had a de

- escalation to decrease the test material concentration to 22500 ppm. During the palatability phase, males that received 30000 ppm and females that received ≥ 22500 ppm failed to maintain consistent body weigh t and food consumption during three day periods of exposure. The effects among females that received ≥ 22500 ppm were considered to be of sufficient severity to preclude the use of these concentrations during subsequent studies, whereas the effects among males that received 30000Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 95 of 126 ppm were not. Therefore, concentrations for the subsequent 28

- day exposure phase were set at 300, 3000, and 30000 ppm in males and 300, 3000, and 15000 ppm in females. Therefore, Beagle dogs were selected from the various palatab ility dosing groups and 2 /sex/dose were administered XR

- 729 acid at 0, 300, 3000, 15000 (females only) or 30000 (males only) ppm (0, 11, 80, 635 mg/kg bw/day in males; 0, 9, 85, 323 mg/kg bw/day in females) for 28 consecutive days for the main study. In ad dition, toxicokinetics were evaluated in blood and urine samples collected at 1, 2, 6 and 16 hours post

- dose on Days 27 and 16 hours after food removal on Day 29, and in the 30000 ppm males on Day 24.
There were no treatment

- related effects on survival, o phthalmoscopic parameters, or gross pathology. Clinical signs of toxi city observed in both high

- dose males were atypical muscle tone in one male and atypical palpebral closure in the other during week 4 of the study. The study authors considered the clinical signs to be non

- adverse since they only occurred during Week 4; however, given the other treatment

- related effects observed, the adversity of these observations cannot be excluded (Canada PMRA/US EPA)

. These clinical signs were not considered to b e treatment

- related by the US EPA , as there were only single findings in the 4th study week and they were not observed in the 90

- or the 1

- year dog studies. The high dose males had lower body weights (↓29% overall) compared to controls and lower dose gro ups and both high dose males and females lost body weight over the duration of the 28

- d ay phase. Body weight loss was more marked in high dose males. The body weight loss was associated with decreased food consumption (↓29

- 100% from controls) in high dose males and females over the duration of the 28

- d ay phase and the effect is considere d to be associated with decreased palatability of the diet. Both males at 635 mg/kg bw/day had increased total and HDL

- cholesterol levels at termination. One of the high dose males (animal #113) had decreased red blood cell, hemoglobin, hematocrit and reticulocyte counts, increased LDL

- cholesterol, increased bilirubin

. The other high

- dose male had increased AST levels. The above alterations may be secondary to the marked d ecrease in food consumption and associated body weight loss in high

- dose males in the palatability phase of the study and during the 28

- day phase. At the high dose level, thymic atrophy (1/sex vs. 0 in controls) were observed. The thymic atrophy was attributed to stress by the study authors; however, no supporting information was submitted. In the absence of evidence to the contrary, this finding was considered to be treatment

- related by Canada PMRA and Australia OCS; this observation was considered to be a secondary effect of treatment by US EPA. At ≥80 mg/kg bw/day in males and ≥323 mg/kg bw/day in females, treatment

- related degeneration and regeneration of renal tubules was observed. Canada PMRA/Australia OCS: The palatability phase of t his study identified dose levels for the main study which used only 2 Beagle dogs/sex/dose with animals selected from the palatability study. Therefore, it is considered that no reliable identification of LOAEL/NOAEL values can be made from the main study, which itself is considered is a dose range finding study for a subsequent 90
- day dog study [PMRA 2226469 , MRIDHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 96 of 126 48557831]. Consequently, a NOAEL/LOAEL has not been identified from this palatability/dose range
- finding study which used the same animals in ea ch phase.
US EPA: A NOAEL and LOAEL were selected for purposes of comparison to the definitive subchronic study. The LOAEL is 80 mg/kg bw/day in males, based on degeneration and regeneration of renal tubules and 323 mg/kg bw/day in females, based on body weight loss, reduced food consumption, thymic atrophy and degeneration and regeneration of renal tubules. The NOAEL in males is 11 mg/kg bw/day and 85 mg/kg bw/day in females.
The t oxicokinetic analys e s conducted after 24 or 27 days of dietary exposure indicated daily systemic dose s (AUC 24 h ) of 10, 79, 331 mg/kg bw /day (male) and 9, 78, and 264 mg/kg bw /day (female) was 16, 185, and 5893 μg h m L - 1 in male and 26, 167, and 1900 μg h m L - 1 in female dogs, respectively. Large variation in the plasma concen trations was observed and was attributed to variability in the feeding pattern amongst the dogs

. In male dogs, the increase in the systemic dose was dose

- proportional up to the mid

- dose level , but became more than dose

- proportional at the high

- dose level

. The more than dose proportional increase in the systemic dose at the highest dose in male dogs is consistent with saturation of elimination of XR

729 acid and was also observed at the highest dose in females to a lesser extent . In female dogs, the daily systemic dose increased proportional to dose up to the mid

- dose level. These data were confounded by the high variability between the blood levels from the two female animals and thus, a definitive statement on the linearity of systemic doses across all do se groups cannot be made based on these data alone. Urinary elimination of XR

- 729 acid was 13, 49 and 53 % in male and 41, 70, and 44 % in female dogs at the low, mid and high doses, respectively. Female dogs were more efficient in eliminating XR

- 729 ac id than male dogs. Based on the large variation in the plasma concentrations it is considered that definitive statements on the proportionality of urinary elimination with administered dose cannot be made from the study results. This study in dogs is acce ptable /nonguideline as a range
- finding study.
870.3150 90
- Day Oral Toxicity

– Dog
In an oral 90

- day dog study (MRID 48557831; PMRA 2226469), XDE

- 729 acid (96.5% a.i. lot no. E2978

- 05

- 01, TSN) was administered to four Beagle dogs/sex/group in the diet at dose levels of 0, 500, 2500 or 12500 ppm (equal to 0, 16.75, 80.64 or 423.81 mg/kg bw/day in males and 0, 18.71, 79.88 or 414.74 mg/kg bw/day in females, respectively).
There were no treatment

- related effects on survival, clinical signs, clinical chemistry or ophthalmoscopic parameters, or gross pathology. At 414.74 mg/kg bw/day, ther e was a treatment

- related decrease in body weights (♂ ↓12

- 16%; ♀ 8

- 10% from controls), body weight gains in males and to a lesser extent females (♂ ↓68%; ♀ 61% from controls). In females, treatment

- related reduced food consumption (↓20% from controls) was observed but was not completely attributable to the marked decrease in body weight gain compared to controls. The decreased body weight gain in males and females at the high dose and decreased food consumption in females at this dose was considered to be a dverse.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 97 of 126 In both sexes at the high

- dose level, there were treatment

- related decreases in erythrocytes (♂ ↓20%, ♀ 23% from controls ), hemoglobin (♂ ↓22%, ♀ 18% from controls ) and/or hematocrit (♂ ↓19%, ♀ 17% from controls ) at Week 13 relative to concurrent c ontrols and pre

- test values. There were slight non

- statistically significant increases in reticulocyte counts (♂ ↓32%, ♀ 35

- 190% from controls ) at Weeks 7 and/or 13 compared to concurrent controls and pre

- test values in males and females. In females, th ese changes were associated with bone marrow hyperplasia and increased myelopoiesis identified in histopathological examinations . One of the high

- dose females had increased band neutrophils and reactive lymphocytes, while the other high

- dose female lacked mature neutrophils, had reduced band neutrophils, as well as increased reactive lymphocytes and monocytes indicative of ineffective myelopoiesis and maturation arrest at band neutrophil stage. Increased liver weights (♂ ↑38%, ♀ 51%) accompanied by extrame dullary hematopoiesis in the liver and spleen was also observed in one of the high

- dose females and was considered to be potentially adverse. An additional target organ of toxicity was the kidney in both sexes. Increased incidences of degeneration and re generation of renal tubules, degeneration of collecting ducts, and focal granulomas in the renal cortex or medulla were observed in both sexes at the high dose

. Additionally, at the high

- dose level, 3 males (#130/10A2918, #131/10A2919, #132/10A2920) and 2 females (#126/10A2934, #127/10A2935) had treatment

- related decreased thymus weights compared to controls. This chan ge in organ weight was accompanied with thymic atrophy upon histological examination in all cases except for male #130. These effects were at tributed to stress by the study authors; however, no supporting information was submitted. In the absence of evidence to the contrary, these findings were considered to be treatment

- related by Canada PMRA and Australia OCS; the US EPA considered this to be a secondary effect of treatment. The LOAEL is 423.81/414.74 mg/kg bw/day in males/females, based on decreased body weights (Canada PMRA/US EPA), decreased body weight gains, reduced food consumption (females), reduced red cell parameters (red blood cells, hemoglobin, hematocrit levels) an d associated bone marrow hyperplasia characterised by an increase in myelopoiesis (females), decreased thymus weights and associated thymic atrophy, renal toxicity (including degeneration and regeneration of renal tubules) and increased liver weight and as sociated extramedullary hematopoiesis of liver and spleen (females). The NOAEL in males is 80.64 mg/kg bw/day and 79.88 mg/kg bw/day in females. Toxicokinetic analysis of XDE

- 729 acid in plasma during week 13 of dietary administration showed that the eli mination half

- life (t 1⁄2 ) of XDE

- 729 was 2.37 hours in males and 4.89 hours in female dogs of the high

- dose group. The elimination t 1⁄2 could not be calculated for the mid

- and low

- dose groups because plasma concentrations at 27 hours were below the limit of quantitation in those groups. The daily systemic doses (AUC 24h ) for males in the low

- , mid

- and high

- dose groups were 41, 183, and 2917 μg h/ml, respectively. The AUC 24h for females in the low

- , mid

and high

- dose groups were 44, 212 and 3794 μg h/ml, re spectively. When plotted against dose, the AUC 24h values exhibited a linear relationship for the low

- and mid

- dose groups but appeared to become non

- linear at the high dose with a more than dose proportional increase. This nonlinearity was proven statist ically in females but not in males.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 98 of 126 The total dose excreted in 24 hour urine as a percentage of the administered dose was 102.2, 72.6 and 28.6% in males and 105.8, 47.0 and 32.1% in females for the low

- , mid

- and high

- dose groups, respectively. Analysis of XDE

- 729 acid in excreted 24 hour urine across all dose levels showed the renal elimination of XDE

- 729 was sub

- linear at the high dose in male dogs, as proven statistically. In female dogs, XDE

- 729 acid excreted in 24 hour urine showed a sub

linear tren d but, due to high variation among animals, non

- linearity could not be proven by statistical analysis.
The kinetics of XDE

- 729 acid in plasma and urine of male and female dogs during week 13 of dietary exposure appeared to be consistent with kinetics ex pected with the occurrence of saturation of elimination at the high dose. The supraproportional increase in systemic exposure and decrease in urinary elimination at high doses is consistent with observations in rats following a single oral dose of the sal t and methyl forms [Hansen et al., 2010; PMRA 2226446] and in the 28

- day palatability and probe study in dogs [Popke, 2010; PMRA 2226465]. However, kinetic nonlinearity in the daily systemic doses (AUC 24h ) could be proven statistically only for females, and kinetic nonlinearity in the total dose excreted in 24 hour urine could be proven statistically only in males. This subchronic toxicity study in the dogs is classified as acceptable/guideline, and satisfies the guideline requirement for a subchronic or al study (OPPTS 870.3100; OECD 408) in dogs.
870.3200 21/28
- Day Dermal Toxicity

– Rat In a repeat

- dose dermal toxicity study (MRID 48557834, PMRA 2226471) , XDE

- 729 acid ( 95.3% a.i.) suspended in 0.5% aqueous methylcellulose vehicle (dosing volume 4 mL/kg bw) was applied to the shaved skin of 10 F344/DuCrl rats /sex/dose at dose levels of 0, 100, 300 or 1000 mg/kg bw /day, 6 hours/day for 7 days/week during a 28

- day period. The treatment site was semioccluded during exposure.
There were no treatment

- re lated effects on survival, clinical signs, body weights, food consumption, ophthalmoscopic parameters, hematology, clinical chemistry, urinalysis, organ weights, gross pathology or histopathology. The LOAEL was not established, as no treatment
- related ef fects were observed up to a limit dose. The NOAEL is 1000 mg/kg bw /day. This 28 day dermal toxicity study in rats is classified as acceptable /guideline and satisfies the guideline requirement ( OPPTS 870.3200, OECD 410 ) for a 21/28 day repeat

- dose dermal t oxicity study in rats.
870.3465 90
- Day Inhalation

– Rat Study not conducted due to inability to generate acceptable test atmosphere
A.4.2 Prenatal Developmental ToxicityHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 99 of 126 870.3700a Prenatal Developmental Toxicity Study
- Rat In an oral developmental toxicity study (PMRA 2226500, MRID 48557835), gr oups of 26 time

- mated female Crl:CD(SD) rats were administered diets containing 0, 500, 2000, or 8000 ppm XDE

- 729 acid (95.3% a.i., lot no. E2837

- 52, TSN

- 030751

- 0006) on gestation day (GD) 6 through 21 (equal to 0, 34.1, 140, or 526 mg/kg bw /day, respectively ) . In

- life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterat ions. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one hal f of the fetuses were examined for visceral and craniofacial alterations, while skeletal examinations were conducted on the remaining fetuses.
There were no treatment

- related effects on maternal liver weights or gross pathology. Two maternal animals at 52 6 mg/kg bw/day were sacrificed prematurely for humane reasons on gestation days 17 or 18 due to excessive body weight losses, reduced food consumption and decreased fecal output. These mortalities were considered to be treatment

- related. Increased incidenc es of decreased fecal output (4 females) and perinasal red soiling (2 females) were noted at 526 mg/kg bw/day. Decreased body weight (GD 21; 6%), body weight gains (37%) and food consumption (6

- 18%) were observed throughout the study at the high dose level , but most notably during the last few days of gestation. Treatment

- related decreases in gravid uterine weights (10%) and increased relative kidney weights (11%) were also observed at 526 mg/kg bw/day. The maternal LOAEL is 526 mg/kg bw/day based on increased mortality, decreased fecal output and increased perinasal red soiling, decreased body weight, body weight gains and food consumption, decreased gravid uterine weights, and increased relative kidney weights. The maternal NOAEL is 140 mg/kg bw/day.
No treatment

- related external or visceral malformations or variations were observed. There was no evidence of treatment

- related skeletal malformations in any dose group. In the 526 mg/kg/day group, treatment

- related decreased fetal weights (3

- 5%), increa sed total resorptions (incidences: 8, 13, 16, 22), resorptions per dam (0.9 vs. 0.3 in controls) and post

- implantation loss (*2.2, 3.6, 4.5, 6.2%), and increased incidences of delayed ossification of thoracic centra (designated a developmental variation; f etal(litter) incidences: 2(1), 1(1), 2(1), 7(7)) were observed.
T he developmental LOAEL is 526 mg/kg/day based on marginal effects including decreased fetal weights , increased total resorptions, resorptions per dam and post
implantation loss, and increas ed incidences of delayed ossification of thoracic centra (designated a developmental variation). The developmental NOAEL is 140 mg/kg/day. The developmental toxicity study in the rat is as classified acceptable /guideline and satisfies the guideline requir ement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats. Nonguideline Prenatal Developmental Rangefinding Study

– RabbitHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 100 of 126 In a developmental toxicity study (PMRA 2226504, MRID 48557837), g roups of 26 time

- mated female New Zealand Whit e rabbits were administered diets containing 0, 4000, 10000, or 28292 ppm XDE

- 729 acid (95.3%, lot no. E2837

- 52, TSN030751

- 0006) on gestation days (GD) 7

- 28 which corresponded to time

- weighted average doses of 0, 170, 434, or 1094 mg/kg body weight/day, respectively. In

- life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and f oo d consumption. On GD 28, all surviving rabbits were euthanized and examined for gross pathologic al alterations. Liver, kidney s, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external and visceral alterations. The heads were also exami ned for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses. Plasma concentrations of XDE

- 729 acid were measured in pregnant does and their respec tive litters on GD 28 to confirm systemic exposure and evaluate dose

- proportionality.
Maternal toxicity : There were no treatment

- related effects on maternal survival, clinical signs, gross pathology, terminal body weights, kidney, liver or gravid uterus weights, or Caesarean section parameters. Treatment

- related decreases in body weight gains (14%) and food consumption (5

- 20%) were observed during gestation. The maternal LOAEL is 1094 mg/kg bw /day, based on decreased body weight gains and food consumpti on during gestation. The maternal NOAEL is 434 mg/kg bw /day. Developmental toxicity : There were no treatment

- related effects on fetal survival or body weight, and no developmental malformations or variations upon external, visceral, craniofacial or skel etal examination. The developmental LOAEL was not established. The developmental NOAEL is 1094 mg/kg bw /day. The mean terminal plasma concentrations of XDE

- 729 acid in both dam and fetal blood demonstrated systemic exposure. On average across all dose groups, fetal XDE

- 729 acid plasma concentrations were 3.0±0.9

- fold lower than doe plasma concentrations (i.e. fetal plasma concentrations were 26

– 47% of doe plasma concentrations) . Analysis of kinetic dose proportionality was not possible due to high var iability between animals in each dose group, likely attributable to a combination of XDE

- 72 9 acid ’s short plasma half

- life in the rabbit (1.08 h) and maternal feeding patterns at the end of pregnancy. This study is classified as acceptable /guideline and s atisfies the guideline requirement for a Prenatal Developmental Study

– Rabbits; OPPTS 870.3700; OECD 414.

A.4.3 Reproductive Toxicity Nonguideline Dose Range
- Finding Study

– RatHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 101 of 126 In an oral reproductive toxicity probe dose range

- finding study (MRID 48557880; PMRA 2226495), g roups of 12 male and 12 female Crl:CD(SD) rats were given test diets initially formulated to supply XDE

- 729 acid (96.5% a.i., lot no. E2978

- 05

- 01, TSN030751

- 0005) at doses of 0, 50, 250, 750, or 1000 mg /kg bw/day. The time

- weighte d doses were 0, 53.4, 266 or 754 mg/kg bw/day in males and 0, 53.9, 269 or 535 mg/kg bw/day, respectively. Males were fed the test diets for four weeks prior to breeding, continuing throughout breeding, and until necropsy on test day 47. The females were fed the test diets for four weeks prior to breeding, continuing through breeding (up to two weeks), through gestation (three weeks) and until necropsy on lactation day (LD) 14. Effects on gonadal function, mating behavior, conception, development of the c onceptus, parturition and postnatal growth and survival were evaluated. In addition, a gross necropsy and histopathologic al examination of the liver and kidney of adults were conducted. Plasma concentrations of XDE

- 729 acid were measured in adult males o n days 15 and 28, and in adult females and their culled pups on day 4 postpartum to confirm systemic exposure and evaluate dose

- proportionality. Parental toxicity : During the first week of treatment, males and females given a targeted dose of 1000 mg/kg bw /day had lower f oo d consumption, body weights, and fecal output relative to controls, with 4/12 males and 4/12 females losing weight. This level of toxicity exceeded the maximum tolerated dose and was considered excessive for use in subsequent studies. Therefore, the 1000 mg/kg bw /day group was terminated on day 8 with no further collection of data. In the 754/535 mg/kg bw /day M/F mg/kg/day group, 1/12 males and 4/12 females lost weight between days 7

- 14. Therefore, the targeted dose level for both sexes was reduced from targeted 750 to 500 mg/kg bw /day for the final two weeks of the pre

- breeding period. One female continued to lose body weight and was removed from the study for humane reasons on d ay 20. Upon necropsy, this animal exhibited decreased ingesta in the gastrointestinal tract and a decreased amount of adipose tissue grossly and numerous adverse renal histological lesions; therefore, this mortality was considered to treatment

- related. I n the remaining females, gestation body weight and body weight gain was decreased, while there were no effects on f oo d consumption, body weights, or body weight gains in males or lactating females. In parental animals, there were no treatment

- related eff ects on kidney or liver weights. The primary target organ associated with dietary exposure to XDE

- 729 acid was the kidney in males and females given 754/535 mg/kg/day M/F. Treatment

- related histopathological alterations consisted of 1) multifocal hypertr ophy (incidences: ♂ 0, 0, 0, 6; ♀ 0, 0, 0, 10) and hyperplasia (incidences: ♂ 0, 0, 0, 3; ♀ 0, 0, 0, 9) of the epithelial cell lining of the kidney collecting ducts with accompanying nuclear karyomegally, 2) necrosis of individual collecting duct epithelia l cells (incidences: ♂ 0, 0, 0, 2), 3) increased mitotic figures (mitotic alteration) within the collecting ducts (incidences: ♀ 0, 0, 0, 1), and 4) tubular dilatation (incidences: ♀ 0, 0, 0, 5). Lesions were more prominent in females than males at the hi gh

- dose level. There was significant variability in the occurrence and severity of the treatment

- related effects observed in individual animals at the high

- dose level. Reproductive toxicity : No reproductive toxicity was observed. Offspring toxicity : In offspring, there were no treatment

- related effects on clinical signs, litter size or pup body weights.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 102 of 126 Canada PMRA, Australia OCS: Maternal, reproductive and offspring NOAELs and LOAELs were not established in this range
- finding study.
US EPA: NOAE Ls/LOAELs were identified for comparison with the definitive study. Parental systemic : The LOAEL is 754/535 mg/kg bw/day M/F, based on decreased gestation body weight and weight gain, kidney histopathology (multifocal hypertrophy/hyperplasia of collecting duct epithelial cell lining, nuclear karyomegally, necrosis of individual collecting duct epithelial cells, increased number of mitotic figures within collecting duct cells and tubule dilatation with inflammation) . The NOAEL is 255/269 mg/kg bw/day M/F. Re productive : The LOAEL was not determined, as no dose related effects were seen. The NOAEL is 754/536 mg/kg bw/day M/F. Offspring : The LOAEL was not determined, as no dose related effects were seen. The NOAEL is 754/536 mg/kg bw/day M/F. Blood samples coll ected from adult males (days 15 and 28) and females (LD 4) and PND 4 culled pups indicated no deviation from linearity for systemic exposure of the test material. Following the reduction of the high

- dose level, the mean plasma concentration was proportion ately reduced and systemic linearity was maintained as demonstrated by the day 28 plasma concentrations falling on the regression line. In PND 4 culled male and female pups, mean plasma XDE

- 729 concentrations were 9 and 11%, respectively, of the dam plas ma levels. Interpretation of the relative pup plasma concentrations should be limited to a demonstration of exposure as the time elapsed between the last nursing event and pup blood collection is unknown (potentially several hours) and the molecule has a very short plasma half

- life (t1⁄2α = 0.14 h). This reproductive/developmental toxicity screen is classified as acceptable/non

- guideline as a range
- finding study; it was conducted as a dose range

- finding study but does not meet the requirements for a Reprodu ction/Developmental Probe Study

-- Rats (OPPTS 870.3550, OECD 421). 870.3800 Reproduction and Fertility Effects

– Rat
In a n oral two

- generation reproducti ve toxicity study (PMRA 2226496, MRID 48557841), g roups of 27 male and female Crl:CD(SD) rats were administered diets supplying XDE

- 729 acid (95.3% a.i., lot no. E2837

- 52, TSN030751

- 0006) at concentrations equivalent to target doses of supplying 0, 20, 100, or 450 mg XDE

- 729 acid /kg bw /day for ten weeks prior to breeding, and through breeding, gestation and lactation for two generations. The time

- weighted doses for the F 0 generation were 0, 20.7, 104 or 465 mg/kg bw/day in males and 0, 20.5, 103 or 465 mg/kg bw/day in females. The time

- weighted doses for the F 1 generation were 0, 20.3, 101 or 459 mg/kg bw/day in males and 0, 19.5, 97.7 or 443 mg/kg bw/day in females. In

- life parameters included clinical observations, f oo d consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In additio n, post

- mortem evaluations included gross pathology and organ weights in weanlings, and gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 103 of 126 Parental toxicity : In parental animals, there were no treatment

- related effects on survival, estrous cyclicity, sperm parameters, reproductive performance or ovarian follicle counts. At the high

- dose level, treatment

- related clinical signs including decreased quantity and abnormal colour of feces, pe riocular soiling, dehydrated skin, pale mucous membranes and a thin appearance were observed in two females in late gestation and early lactation ; these animals also had decreased body weight gains (7%) and food consumption (10%) from GD 14

- 2 1. Treatment

r elated effects at the high

- dose level included increased kidney weights (absolute/relative 10%/7%) in females and renal histopathology in both sexes. Renal toxicity was characterized by increased incidences of collecting duct epithelial cell hypertrophy (i ncidences: F 0 ♂ 1, 0, 0, 10, ♀ 0, 0, 0, 17; F 1 ♂ 1, 0, 1, 16, ♀ 0, 0, 0, 19) and hyperplasia (incidences: F 0 ♂ 0, 0, 0, 9, ♀ 0, 0, 0, 9; F 1 ♂ 1, 0, 1, 12, ♀ 0, 0, 0, 8) , hyperplasia of the pelvic epithelium (incidences: F 1 ♂ 0, 0, 0, 6) and increased mitot ic figures (incidences: F 0 ♂ 0, 0, 0, 2, ♀ 0, 0, 0, 1; F 1 ♂ 0, 0, 0, 6) in the collecting duct epithelium . Other adverse histological observations included increased incidences of mineralization of the aorta (incidences: F 1 ♀ 1, 0, 0, 8) , renal arteries ( incidences: F 0 ♀ 0, 0, 1, 2; F 1 ♀ 0, 0, 0, 4) , renal tubular basal lamina (incidences: F 1 ♀ 0, 0, 0, 5) and mammary gland artery (incidences: F 1 ♀ 0, 0, 0, 2). The parental LOAEL is 465/465 mg/kg bw/day M/F, based on renal histopathological observations (collecting duct epithelial cell hypertrophy and hyperplasia, hyperplasia of pelvic epithelium, mitotic figures of the collecting duct epithelium) in both sexes; clinical signs, decreased body weight gains and food consumption during late gestation/early lactation, increased kidney weights, increased incidences of mineralization of the aorta, renal arteries, renal tubular basal lamina and mammary gland artery in females. The parental NOAEL is 104/103 mg/kg bw/day M/F. Reproductive toxicity : There was no e vidence of reproductive toxicity. Offspring toxicity: In offspring, there were no adverse effects on viability, clinical signs, litter parameters, body weights, sexual maturation, organ weights or gross pathology. The reproductive and offspring LOAELs were not established (>443 mg/kg bw/day) . The reproductive and offspring NOAELs are 443 mg/kg bw /day. This study is classified as acceptable /guideline and satisfies the guideline requirement for a Multi

- generation Reproduction Study

-- Rats (OPPTS 870.380 0, OECD 416).
A.4.4 Chronic Toxicity 870.4100a (870.4300) Chronic Toxicity

– Rat
In a 2

- year combined chronic/oncogenicity study (PMRA 2226483, MRID 48557842), groups of 60 male and 60 female F344/DuCrl rats were fed diets formulated to provide 0, 20, 100, 400, 625 (males only) or 750 (females only) mg XDE

- 729 acid ( 95.3% a.i., lot no. E2837

- 52, TSN030751

- 0006) /kg body weight/day. These dietary doses corresponded to time

- weighted average doses of 0, 20.2, 101, 404 and 633 mg/kg bw/day for males and 0, 20.3, 102 or 407 mg/kg bw/day, respectively for females. Females given 750 mg/kg bw/day were terminated onHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 104 of 126 test day 518 due to excessive mortality (56%), indicative of exceeding a maximum tolerated dose (MTD); these animals were subsequently removed f rom the study and no histopathological assessments were conducted. Ten rats/sex/dose level were necropsied after one year (chronic toxicity group) and the remaining 50 rats/sex/dose level were maintained on their respective diets for up to two years. Anim als were evaluated by cage

- side examinations, clinical observations, detailed clinical observations, body weight, food consumption and food efficiency. Ophthalmic examinations were conducted on all rats, pre

- study and all surviving rats prior to the sched uled necropsies. Clinical pathology examinations (hematology, coagulation, clinical chemistry, electrolytes, and urinalysis) were conducted at regular intervals throughout the study. Toxicokinetic analysis was conducted on blood and urine samples from 4 rats/sex/dose at 6 and 12 months post

- dosing. All rats had a complete necropsy with weights of selected organs recorded at the scheduled necropsies. Histopathological examination was performed on all control and high

- dose level rats and all rats that die d spontaneously or were euthanized due to their moribund condition. Histopathological examinations were not conducted on the female 633 mg/kg bw/day dose group due to early termination from the study. Histopathological examination of rats from the low

- a nd intermediate

- dose levels of the 12

- month chronic to xicity group was limited to the target organ (kidney) and relevant gross lesions. Histopathological examination of survivors from the low

- and intermediate

- dose levels of the 24

- month oncogenicity group was limited to the kidneys and relevant gross lesions for both sexes, and the urinary bladder, adrenal glands, and mesenteric tissues for males. The cumulative mortality of males given 633 mg/kg bw /day at 24 months was higher and statistically significant as compared to controls . The m ortality rates at the end of the study were 32, 44, 44, 34 and 62% in males and 16, 10, 28 and 30% in females in the specified dose groups, respectively . The higher mortality w as attributed to treatment

- related toxic effects in the kidneys. The increased mortality was not due to tumo u rs, which indicate s that this dose level had exceeded an MTD. An MTD was achieved for females given 407 mg/kg bw/day, based on treatment

- related degeneration with regeneration of renal tubules and necrosis of individual epithelial cells lining the collecting ducts was observed in females at this dose level. There were no treatment

- related effects on survival in the remaining dose groups.
At 633/7 66 mg/kg bw/day M/F, in addition to decreased survival, treatment

- related decreases in body weight and/or body weight gains, as well as a roughened kidney surface (24 mo nths) were observed in both sexes. In males, increased incidences of mineralization of the pelvic epithelium in kidney , increases in the incidence of very slight necrosis of individual epithelial cells of the tubules and atrophy of mesenteric adipose tissue (secondary to decreased body weights) were also observed. Treatment

- related increased blood urea nitrogen levels , necrosis of collecting duct epithelial cells of the kidney and vacuolization of the zona glomerulosa of the adrenal glands were noted at doses greater than the MTD in females.
There were no adverse effects on clinical observations, f oo d consumption, f

oo d efficiency, ophthalmologic al observations or hematology parameters in any of the dose groups

. At 404/407 and 633/766 mg/kg bw/day M/F in males and females, treatment

- related renal effects consisted of increases in urina ry volume (53

- 270%) , decreased urine specific gravity (0.017

- 0.045) , increased kidney weight s (absolute 8%, relative 12

- 14%) , and increased incidences of renal histopathology [ hypertrophy of renal collecting duct epithelium (incidences: ♂ 27, 29, 30,Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 105 of 126 48, 4 7, ♀ 8, 5, 11, 49) , increased numbers of mitotic figures in collecting duct epithelial cells ( males , 12 mo nths only ; incidences: 0, 0, 0, 3, 1 ), chronic interstitial inflammation of medulla (incidences: ♂ 14, 18, 17, 37, 38, ♀ 9, 3, 14, 40) , hyperplasia of pelvic epithelium ( females, 12 mo nths, incidences: 0, 0, 1, 6, 9 ), vacuolization of collecting duct epithelium in the papilla (12 months, incidences: ♂ 0, 0, 0, 4, 4; ♀ 0, 0, 0, 9, 10) , slight necrosis of collecting duct epithelium and individual tubular epithelial cells (24 mo nths; incidences: ♂ 0, 1, 0, 1, 21; ♀ 0, 0, 1, 6). In males at 404 and 633 mg/kg bw/day, decreased terminal body weights (5

- 7%), and increased incidences of hypertrophy of the zona glomerulosa of the adrenal glands (incidences: 0, 0 , 0, 2, 26) , presence of calculi in the renal pelvis of the kidney (incidences: 0, 0, 0, 1, 2) , degeneration and regeneration of renal tubules (incidences: 0, 1, 0, 4, 26) , bladder effects [ extensive hyperplasia of the transitional epithelium (incidences: 0, 1, 0, 3, 7) , subacute to chronic inflammation in the submucosa beneath the hyperplastic epithelium (incidences: 0, 0, 0, 3, 7) , microscopic calculi in the lumen of the urinary bladder (incidences: 0, 0, 0, 1, 2)] were also observed. In females, increase s in chronic progressive glomerulonephropathy (1 2 mo nths only; incidences: 2, 2, 4, 6, 10) and chronic interstitial inflammation (incidences: 0, 0, 0, 3, 7) were noted in the kidney s. At 102 mg/kg bw/day, the only treatment

- related effect was a statistically identified increase in hyperplasi a of pelvic epithelium of the kidney (incidences: 8, 5, 22, 43) in females. The LOAEL in males is 404 mg/kg bw /day, based on increased urinary vol ume, decreased urine specific gravity, decreased terminal body weights and decreased body weight gain, increased kidney weight s , adverse renal histopathology findings, hypertrophy of the zona glomerulosa of the adrenal glands, presence of calculi in the re nal pelvis of the kidney, degeneration and regeneration of renal tubules and bladder histopathology (including calculi). The LOAEL in females is 102 mg/kg bw/day, based on hyperplasia of pelvic epithelium of the kidney . The male NOAEL is 101 mg/kg bw/day a nd the female NOAEL is 20.3 mg/kg bw/day. There was no evidence of oncogenicity. The MTD was achieved at 404/407 mg/kg bw/day M/F based on treatment

- related renal toxicity.
Plasma levels of XDE

- 729 acid (AUC 24h ) at 6 and 12 months of exposure, when plot ted against dose, were linear, with the exception of males at 6 months. Plasma levels in males at 6 months were subli near at the high dose (749 mg/kg/day). Urine levels of XDE

- 729 acid at 6 and 12 months were also linear, when plotted against dose

. Beca use terminal plasma levels (samples taken at the time of necropsy) were either very low or non

- quantifiable (below the lower limit of quantitation), t 1/2 values could not b e calculated for most animals. However, t he average t 1/2 from those that could be calculated was 3. 3 h ours
. At 6 months of exposure, most (approximately 72%, on average) of the administered dose of XDE

- 729 acid was excreted in the urine. At 6 months in females there appeared to be some saturation of urinary elimination at the high d ose with 85, 96, 57 and 55% of the administered dose excreted in 24 hour urine at 20, 97, 425 and 832 mg/kg bw/day, respectively. However, this trend was not apparent in males. At 12 months, there was no apparent trend and the fraction of the administered dose excreted in the urine at 24 hours in of males and females had declined to approximately 41%, on average.Halauxifen

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Page 106 of 126 This study is classified as acceptable/guideline and satisfies the guideline requirement for a Combined chronic/ carcinogenicity [feeding] F344/ DuCrl rats (OPPTS 870.4300; OECD 453). 870.4100b Chronic Toxicity

– Dog
In a 1

- year dog study (PMRA 2226470 , MRID 48557832) , XDE

- 729 (95.3% a.i., lot no. 2857

- 32) was administered to 4 Beagle dogs/sex/group in the diet at dose levels of 0, 500, 2500 or 10000 ppm. Due to the body weight losses of females in the high dose group, the dietary concentration was reduced to 5000 ppm on Day 47 in the females only. The test material intake averaged 13.90, 82.42, and 354.75 mg/kg/day in males and 16.72, 89.85 and 219.80 mg/kg/day in females at the low

- , mid

- and high

- dose respectively.
There were no treatment

- related effects on survival , clinical signs, food consumption, food efficiency, hematology, clinical chemistry, urinalysis or ophthalmoscopic parameters, organ weights or gross pathology. Following 6 weeks of treatment, the mean body weight in high

- dose females was 9% lower in comp arison with the Week

- 1 mean body weight value and body weight losses were observed. The changes in female body weight and body weight gains were considered to be treatment

related but of limited toxicological significance, since recovery was observed aft er the adjustment to the dietary concentration on Day 47 and overall body weight and body weight gains were comparable to controls.
Treatment

- related renal toxicity (graded very slight) consisting of degeneration with regeneration of the renal proximal a nd distal tubules (incidences ♂ 0, 0, 0, 1, ♀ 0, 0, 1, 1) , degeneration of collecting ducts (incidences ♂ 0, 0, 0, 1, ♀ 0, 0, 1, 0) and multifocal glomerulosclerosis (incidences ♂ 0, 0, 0, 2, ♀ 0, 0, 1, 0) was observed in mid

- and high

- dose females and in the high

- dose males upon histological examination. In males, treatment

- related granulomas (incidences ♂ 0, 0, 0, 1) and multifocal fibrosis (incidences ♂ 0, 0, 0, 1) were observed in the renal cortex of the kidneys at the high dose

- level.
The LOAEL is 89 .85 mg/kg bw/day, based on degeneration with regeneration of the renal proximal and distal tubules, degeneration of collecting ducts and multifocal glomerulosclerosis in females. The NOAEL in females is 16.72 mg/kg bw/day. The LOAEL is 354.75 mg/kg bw/day in males, based on degeneration with regeneration of the renal proximal and distal tubules, degeneration of collecting ducts, multifocal glomerulosclerosis, and granulomas and fibrosis in the renal cortex. The NOAEL in males is 82.42 mg/kg bw/day.
XDE

- 729 was present in approximately 50% of plasma, and all urine samples from treated animals , at levels above the analytical lower limit of quantitation. Based on the AUC 24h values, XDE

- 729 exhibited supralinear kinetics at the high dose (10,000 ppm) in male dogs after 13 weeks of exposure. The AUC 24h values also appeared to be supralinear in male and female dogs of the high dose group at 26 weeks of exposure, but this nonlinearity was not statistically significant. At 52 weeks of exposure, there were too few plasma samples with quantifiable levels of XDE

- 729 to assess kinetic linearity across doses. Terminal plasma levels (after 16Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 107 of 126 hours of fasting) were below the lower limit of quantitation, consistent with a relatively short elimination half

- life. On average, males excreted 40% of the dose intact in the urine and females excreted 55% of the dose intact in urine. The total amount of XDE

- 729 excreted in the urine over 24 hours exhibited a linear relationship between all three doses in females and bet ween the 500 and 2500 ppm doses in males. Urine levels appeared to be sublinear at 10,000 ppm in males at 13, 26 and 52 weeks of exposure, though this nonlinearity was not found to be statistically significant. This study in dogs is classified as accepta ble /guideline and satisfies the guideline requirement for a 1

- year oral toxicity study in dogs (OPPTS 870.3150; OECD 409).
A.4.5 Carcinogenicity 870.4200a Carcinogenicity Study
- rat
See 870.4100 870.4200b Carcinogenicity (feeding)
- Mouse In an oncogenicity study (MRID 48557843, PMRA 2226485), g roups of 50 male Crl:CD1(ICR) mice were fed diets formulated to provide 0, 50, 250, or 750 mg/kg body weight/day (mg/kg/day) and groups of 50 female Crl:CD1(ICR) mice were fed diets formulated to pro vide 0, 50, 250, or 1000 mg/kg bw /day XDE

- 729 acid (95.3% a.i., lot no. E2837

- 52, TSN030751

- 0006) for up to 18 months to evaluate the potential for oncogenicity. These dietary concentrations corresponded to time

- weighted average doses of 0, 50, 251, or 75 1 mg/kg bw /day for males, and 0, 50.1, 251, or 1004 mg/kg bw /day for females, respectively. A nimals were evaluated by cage side observations, clinical observations, detailed clinical examinations, body weights, and feed consumption. Ophthalmic examination s were conducted pre

- exposure and prior to necropsy. Toxicokinetic analysis was conducted on blood and urine samples from 4 mice/sex/dose at 6 and 12 months. All mice had a complete necropsy examination with total white blood cell (WBC) and differential WBC counts and weights of selected organs recorded at the scheduled necropsy. An extensive set of organs was examined histopathologically from all control and high

- dose group mice, as well as all mice that died or were euthanized in a moribund condition. Histopathologic al examination was also conducted on target organs, selected major organs and all gross lesions of mice from the low

- and intermediate

- dose groups from the terminal necropsy.
There were no adverse effects on clinical observations (includin g detailed ranked assessments) , food consumption, food efficiency, ophthalmological observations , hematology parameters (white blood cell count, leukocyte differential count), or incidence of palpable masses in any of the dose groups. At 251 mg/kg bw /day and above , treatment

- related moderate subacute to chronic inflammation (incidence: 0, 4, 3, 27) and gross and microscopic calculi in the lumen (incidence: 0, 0, 2, 10) were observed in the kidneys of males. In females, the only treatment

- related effects wereHalauxifen

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Page 108 of 126 decreased body weight gains (8

- 9%) and increased hypertrophy of the intercalated cells in kidneys (incidence: 1, 1, 8, 36)

. The highest dose tested in male mice ( 751 mg/kg bw /day) was considered to be in excess of the maximum tolerated dose. The mor tality rate at study termination was 4 6% at the highest dose tested and was statistically identified and attributed to the high prevalence of treatment

- related renal and urinary bladder toxicity in deceased males . There were no treatment

- related effects on survival in the remaining dose groups. At 751 mg/kg bw /day in males, treatment

- related increases in mortality due to renal and urinary bladder lesions were observed along with decreased kidney weight (absolute 8%, relative 7%) , urinary bladder toxicity [ thickened bladder wall (incidences: 0, 2, 0, 7) , hyperplasia of the transitional epithelium lining of the mucosa (incidences 0, 1, 0, 7) , increased numbers of mitotic figures within the epithelium (incidences: 0, 0, 0, 10) , ulceration of the epithelium (i ncidences: 0, 0, 0, 2) , necrosis of individual transitional epithelial cells (incidences: 0, 1, 0, 11) ; decreased aggregates of mononuclear cells secondary to inflammation (incidences: 31, 31, 32, 11)] and renal toxicity [ degeneration with regeneration of cortical tubular epithelium (incidences: 0, 0, 0, 12) , tubular dilatation (incidences: 1, 1, 3, 14) , hypertrophy of the intercalated cells of the collecting ducts (incidences: 2, 0, 0, 39)]

. There were no treatment

- related effects in females at 1004 mg/kg bw/day. The LOAEL in both sexes is 251 mg/kg bw /day, based treatment
- related moderate subacute to chronic inflammation and gross and microscopic calculi in the lumen of the kidneys of males and decreased body weight gains and increased hypertrophy of the intercalated cells in kidneys of females. The NOAEL in males and females is 50 mg/kg bw /day. There was no evidence of oncogenicity. The MTD was exceeded in males at 751 mg/kg bw /day M/F based on treatment

- related mortalities attributed to renal and urinar y bladder toxicity. A limit dose (1004 mg/kg bw/day) was tested in females. The plasma concentrations of XDE

- 729 acid were dose proportional (linear) in females at 6 and 12 months and in males at 12 months. Less than dose

- proportional plasma concentratio ns of XDE

- 729 acid were observed (sublinear) at the high dose in males at 6 months. XDE

- 729 concentrations in urine were dose

- proportional in both male and female mice at 6 and 12 months of dietary exposure. These data are consistent with data from previ ous studies in mouse and rat, in which plasma and urine concentrations of XDE

- 729 acid were usually dose

- proportional, but occasionally sublinear at high doses. Though not statistically significant, visually there appears to be a trend toward sublinearity in plasma concentrations at high doses in the current and previous studies. These data suggest that the high dose of ~750 mg/kg bw /day may be approaching a dose at which nonlinear kinetics would be consistently observed. This carcinogenicity study in mice is classified as acceptable /guideline and satisfies the guideline requirement for a carcinogenicity study (OPPTS 870.4200; OECD 451 ) in mice.
A.4.6 MutagenicityHalauxifen

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Page 109 of 126 Gene Mutation Guideline 870.5100, bacterial reverse mutation test (preincubation assay) MRID 48557844 Acceptable/guideline 0, 50, 10, 500, 1 5 00, or 5000 μg/plate +/

- S9 Negative in Salmonella typhimurium strains TA 1535, TA1537 , TA98, and TA100 and Esc h erichia coli Wp2 uvrA up to the limit concentration with no evidence of compound insolubil ity or cytotoxicity

. Guideline 870.5300, in vitro mammalian cell forward gene mutation, Chinese hamster ovary cells, HGPRT locus MRID 48557848 Acceptable/guideline 0, 187.5, 375, 750, 1500, 2000, 2500, 3000 or 3400 μg/plate +/

- S9 Negative for induced mutant colonies at the HGPRT locus up to precipitating and cytotoxic concentrations.

Cytogenetics
Guideline 870.5375, in vitro mammalian cell chromosomal aberration assay, primary rat lymphocytes, SD rat MRID 48557852 Acceptable/guideline 0, 800 , 15 00 or 2000 μg/mL in the absence of mammalian metabolic activation (S9) ; 0, 40 0, 800 or 1500 μg/mL in the absence of mammalian metabolic activation Negative for induction of chromosomal aberrations up to cytotoxic concentrations.

Guideline 870.5395, in vivo mammalian micronucleus assay, CD1 mouse MRID 48557856 Acceptable/guideline 0, 500, 1000 or 2000 mg/kg Negative for increased frequency of micronucleated reticulocytes in peripheral blood up to a dose causing systemic toxicity.

A.4.7 Neurotoxicity 870.6100 Delayed Neurotoxicity Study
- Hen Study not required 870.6200 Acute Neurotoxicity Screening Battery In an acute oral neurotoxicity study (MRID 48557857; PMRA 2226505), t en male and ten female F344/DuCrl rats per group were given a single, oral gavage dose of 0, 250, 750, or 2000 mg XDE

729 technical /kg body weight (acid form, 95.3% a.i., Lot no. E2837

- 52, TSN030751

- 0006) . Body weights were recorded and a functional observational battery (FOB) and an auto mated test of motor activity were conducted pre

- exposure (baseline), the day of dosing (day 1), day 8, and day 15. The FOB included hand

- held and open

- field observations as well as measurements of grip performance, landing foot splay and rectal temperature . Clinical observations were conducted on days 2, 3, and 4 , and body weights were also recorded on day 2 . At the end of the study , on day 16, five rats/sex/dose level were perfused in situ for histopathologic evaluation of the central and peripheral nervou s systems, which was conducted on the control and high

- dose groups. Positive control data demonstrating the proficiency of the laboratory personnel in conducting neurobehavioral assessments were provided.Halauxifen

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Page 110 of 126 Treatment with XDE

- 729 did not affect clinical obs ervations (days 2

- 4), rectal temperature, grip performance, landing foot splay, or motor activity in males or females, at any dose level tested. There was a slight, treatment

- related decrease in the body weight of males and females given 750 or 2000 mg/kg bw on days 2, 8 and/or 15 when compared to controls , with the greatest decrease seen in males at 2000 mg/kg bw on Day 2 (

- 7.8%)

. This effect was not considered adverse due to the small magnitude of the change at all time points (<10%). At the end of the study, body weight gain s of males given 750 or 2000 mg/kg bw /day were lower (

- 9%/

- 11%) when compared to controls . Body weight gains in females were not affected by treatment. There was a slight decrease in the level of activity during the open

- field evalua tion of the FOB on day 1 in male rats only given 750 or 2000 mg/kg bw (2.3 at both doses versus 2.9 in controls) , which was considered an equivocal effect due to the lack of an effect on other related neurobehavioral endpoints including motor activity and absence of any treatment related neuropathological findings

. The results of the neuropathologic evaluation indicated that XDE

- 729 had no effect on the central or peripheral nervous system. Canada PMRA, and Australia OCS: Based on the decrease in body weight in males on Day 2, the NOAEL for F344/DuCrl male rats dosed with XDE
- 729 was 750 mg/kg bw. In females, no adverse effects were observed up to the highest dose tested, therefore the NOAEL was set at 2000 mg/kg bw. There was no evidence of neurotoxicity up to the highest dose tested.
US EPA : The neurotoxicity and systemic toxicity NOAELs are 2000 mg/kg bw (HDT and limit dose) for both males and females. LOAELs for neurotoxicity and systemic toxicity were not identified (>2000 mg/kg bw). This study is classified as acceptable/guideline and satisfies the guideline requirement for an acute neurobehavioral screening study in the rat ( OPPTS 870.6200; OECD 424). 870.6200 Subchronic Neurotoxicity Screening Battery
In a subchronic neurotoxicity study (MRID 48557858; PMRA 2226506), XDE

- 729 (acid form, 95.3% a.i., lot no. E2837

- 52, TSN030751

- 0006) was administered in the diet at levels equivalent to target doses of 0, 50, 250, and 750 mg/kg body weight/day in male and female F344/DuCrl rats for at least 13 weeks. The time

- weighed average dosages were: 0, 52.5/51.2, 260/256, or 774/772 mg/kg body weight/day in M/F. Ten rats/sex/dose were evaluated pre

- exposure and at 2, 4, 8, and 13 weeks of exposure using a functional observational battery (FOB), determinations of grip performance, rectal temperature, landing foot splay, and an automated test of motor activity. Weekly clinical observations, body weights and food cons umption were also evaluated, and ophthalmic examinations were conducted pre

- and post

- exposure. At the end of the study, five rats/sex/dose level were perfused for histopathologic evaluation of the central and peripheral nervous systems, which was conducte d on the control and high

- dose groups. Positive control data were provided that demonstrated proficiency of the testing laboratory personnel.
Treatment with XDE

- 729 did not affect FOB observations, grip performance, rectal temperature, landing foot splay, motor activity, ophthalmic examinations, or clinicalHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 111 of 126 observations in males or females at any dose level tested. At the end of the study, there was a treatment

- related decrease in the body weight (7%) and body weight gain (12%) of males given 750 mg/kg bw / day when compared to controls, which correlated with a slight, treatment

- related decrease in feed consumption across the study in this group. There were no treatment

- related gross or histopathologic observations in the central or peripheral nervous systems of rats administered XDE

- 729.
Canada PMRA : The no

- observed

- adverse

- effect level (NO A EL) for systemic toxicity was 250 mg/kg bw /day based on the decrease in body weight of males at 750 mg/kg bw/day. T he NO A EL for females was 750 mg/kg bw /day ; a LOAEL was not identified

. There was no evidence of neurotoxicity.
US EPA and Australia OCS: The no
- observed
- adverse
- effect level (NOAEL) for both neurotoxicity and systemic toxicity was 750 mg/kg bw/day (HDT) in males and females. The decrease in body weight of ma les at 750 mg/kg bw/day was not considered biologically significant (<10%). LOAELs for neurotoxicity and systemic toxicity were not identified in this study. This study is classified as Acceptable/guideline and satisfies the guideline requirement for a subchronic oral neurobehavioral screening study in the rat ( OPPTS 870.6200; OECD 424). 870.6300 Developmental Neurotoxicity Study Study not required
A.4.8 Metabolism Nonguideline Metabolism Probe Study

– Rat and Mouse
In a metabolism probe study (MRID 48557859, PMRA 2226444), absorption, distribution, metabolism and elimination (ADME) of uniformly labeled 14 C

- XR

- 729 acid (radiochemical purity 96.6%, lot no. INV 030887

- 0001) was determined in Fischer 344 rats (2/sex) and CD1 mice (4/sex) after adm inistration of a single oral dose at 100 mg/kg bw . Time

- course blood (plasma and RBC) and excreta were collected from rats and time

- course excreta only were collected from mice for up to 72 hours post

- dosing . The urine samples collected during the first 12 hours post

- dosing were analyzed for parent compound and metabolite(s). Additionally, two rats (1 /sex ) and four mice (2 /sex ) were orally dosed with 14 C

- XR

- 729 acid at 100 mg/kg bw and plasma was collected at the peak plasma concentration (t max ; 0.5 hour po st

- dosing estimated from rats ) and analyzed for parent and metabolite(s). The orally administered 14 C

- XR

- 729 acid was rapidly absorbed from the GI tract both by the rats and mice with a t max of approximately 0.5 hours and eliminated mainly in the urine. The rate of absorption of 14 C

- XR

- 729

- derived radioactivity from the GI tract was same for both male and female rats. Total urinary elimination accounted for 65

- 75% of the dose in rats and 65

- 66% of the dose in mice, most of which occurred within the first 12 hours after dosing. Fecal elimination accounted for 12

- 21% in rats and 19

- 20% in mice. Elimination of radioactivity fromHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 112 of 126 plasma was bi

- exponential in the rat with an initial half

- life ( t 1⁄2  ) of 0.14 hours indicating rapid distribution to tissues and ter minal elimination half

- life ( t 1⁄2  ) ranging from 3.37 (male) to 3.67 (female) hours. Elimination of 14 C

- XR

- 729

- derived radioactivity from red blood cells ( RBC ) was mono
- exponential with a half

- life ( t 1⁄2 ) of 0.84 (male) and 0.77 (female) hours.
In the 12

- hour urine s amples , eight peaks were detected, but only one peak was found representing >5% of the dose and was identified as XR

- 729 acid ( parent ) . Four metabolites were tentatively identified: the glucuronide conjugate of O

- demethyl XR

- 729, the sulfate conjugate of O

- demethyl XR

- 729, O

- demethyl XR

- 729, and acyl

- glucuronide conjugate of parent XR

- 729. The metabolic profile looked very similar between sexes and species with only slight changes in the percentages of the peaks. Unchanged XR

- 729 acid was fou nd as the major component in all C max plasma extracts and the acyl glucuronide conjugate of XR

- 729 acid was also found in the male mouse plasma extract, representing approximately 7.5% of the recovered radioactivity

. In summary, XR

- 729 acid is rapidly abs orbed from the GI tract of rats and quickly eliminated from the male and female rat and mouse mainly in urine within the first 12 hours after dosing

. The vast majority of the radioactivity in the urine and plasma of rats and mice was unchanged XR

- 729 acid

. This study is classified as acceptable/nonguideline. It was performed as a probe for definitive general metabolism study and was not conducted to satisfy the guideline requirement for a metabolism study (OPPTS 870.7485, OECD 417) in the rat or mouse. Nonguideline Metabolism

– Dog
In a non

- guideline metabolism study (MRID 48557870; PMRA 2226445), t hree male and three female beagle dogs were administered 50 mg/kg bw 14 C

- XDE

- 729 (96.8%; Lot# DE3

- 102069, INV# INV

- 031412

- 0003), formulated as the sodium salt, via oral gavage. Time

- course plasma and excreta were collected up to 168 hours (7 days) post

- dosing to determine absorption, metabolism, and elimination (AME) of this test material. Urine and fecal samples collected at 168 hours post

dosing and plas ma samples collected at C max and 1⁄2C max were analyzed to characterize and quantify XDE

- 729 acid (parent) and metabolites.
Plasma C max was achieved slightly earlier in females than male dogs (mean t max : 0.7 vs. 1.3 h) and was also slightly higher in female dogs (143 vs. 118 μg

- Na eq./g). The vast majority of the 14 C

- radioactivity cleared from dog plasma by 6 hours post

- dosing. Elimination of radioactivity from the plasma was biphasic, with initial half

- life (t 1⁄2α ) of 1 h indicating rapid distribution to ti ssues and terminal elimination half

- life (t 1⁄2β ) of 9

- 12 h. XDE

- 729 acid represented the major radiolabeled component in plasma determined by LC/MS

- MS (analysis of non

- radiolabel) and liquid scintillation counting. These data are consistent with metabolite profile results obtained by HPLC

- RAM analysis of selected C max plasma samples indicating XDE

- 729 acid as the major component in the plasma of dogs. Comparison of pharmacokinetic parameters with rats showed that the dose corrected plasma AUC 24 in dogs was 2 to 4

- fold higher than rats administered 10 and 100 mg/kg bw of 14 C

- XDE

- 729 sodium salt indicating higher systemic exposure to 14 C

XDE

- 729 acid in dogs following a single oral dose. Analysis of excreta showed that approximately 80% of the dose was excrete d in the urine in 168 h, with the largest fraction excreted within 12 h. Fecal elimination accounted for ~10% (males)Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 113 of 126 or ~14% (females) of the dose, most of which were excreted within 48 hours post

- dosing. In total, 95

- 98% of the dose was recovered from excreta and cage wash within 168 hours post

dosing demonstrating adequate mass balance.
XDE

- 729 acid was the major radio

- labeled component in urine/rinse (77

- 79% of the dose) and was present in feces at levels higher than or similar to those of other iden tified metabolites. The primary metabolites identified in urine, feces, or plasma included O

- demethyl XDE

- 729 and the sulfate and glucose conjugates of O

- demethyl XDE

- 729, hydroxy XDE

- 729, and an acyl glucuronide conjugate of XDE

- 729. In summary, animals dosed by oral gavage with XDE

- 729 sodium salt were systemically exposed to XDE

- 729 acid (and associated metabolites), as assessed by radioactivity and HPLC in plasma samples. Absorption of the dose appeared to be fairly rapid (t max of 0.5 to 1 hour post

dosing) and complete (~80% of the dose was excreted in urine and only 10

- 14% in feces at 168 hours post

- dosing). Elimination from plasma was biphasic and fairly rapid (t 1⁄2α of 1 h and t 1⁄2β of 9

- 12h). Excretion of the dose was fairly rapid; the largest fracti on of the dose was recovered from urine collected at 12 hours post

- dosing and most of the dose excreted in feces was recovered by 48 h after oral gavage. Parent compound generally represented the major radioactive component analyzed in plasma and excreta s amples. This study is classified as Acceptable/non

- guideline. It was conducted to provide additional information on the metabolism of XDE

- 729, namely to evaluate the potential for renal saturation of elimination. The study was not submitted to satisfy the guideline requirement for a general metabolism study (OPPTS 870.7485; OECD 417).
870.7485 Metabolism
- Rat In a rat metabolism study (MRID 48557860, PMRA 2226446), groups of four male and female F344/DuCrl rats were administered 14 C

- XDE

- 729 ( 14 C

- (UL

- phenyl)

- XDE

- 729 [Lot# XS9

- 100040

- 68, 97.2%] or 14 C

- (2,6

- pyridine)

- XDE

- 729 [Lot# XS9

- 100040

- 69, 97.3%]), as the so dium salt, following oral (single or multiple dose of 10 mg/kg bw; single dose of 750 mg/kg bw) and intravenous (IV) exposure (single 10 mg/kg bw). In addition, absorption, distribution, metabolism, and excretion (ADME) of 14 C

- XDE

- 729 methyl (X11393728, 97 .4%) (the methyl ester form of XDE

- 729; 10 mg/kg bw) was also determined in male and female F344/DuCrl rats (n=4/sex) to determine the bioequivalence between the acid (as sodium salt) and methyl ester forms of XDE

- 729. Animals were monitored and excreta co llected for 168 hours (7 days) post

dosing to determine ADME of the test compounds. Orally administered XDE

- 729, as the sodium salt, was rapidly absorbed without any apparent lag time (t max 0.1

- 0.2 hours and 0.3

- 1.8 hours following low oral single doses a nd a high oral single dose, respectively). The t max for the low oral XDE

- 729 methyl group was slightly longer (0.44

- 0.50 hours) compared to the low oral XDE

- 729, as the sodium salt. The percent absorption of the orally administered dose sodium salt in all low oral groups (single 10 mg/kg bw, multiple 10 mg/kg bw, single 10 mg/kg bw pyridine

- ring label) was at least 77

- 93%, based on recovery in urine and non

- GI tissues and was comparable for low oral XDE

- 729 methyl dose group (82%). The percent absorption es timated from urine and non

- GI tissue was slightly lower following theHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 114 of 126 high single oral dose of 14 C

- XDE

- 729 sodium salt (69

- 73%). Total recovery of radioactivity from all the animals averaged 102 ± 3 and 99 ± 3% in the oral and IV dose groups, respectively. Absolute oral bioavailability, calculated from comparison of the plasma AUC data for the low oral and IV dose groups ( 14 C

- (UL

- phenyl)

- XDE

- 729 sodium salt), was ~100% for both female and male rats. The orally absorbed dose was rapidly excreted in urine (68

- 92% of the administered dose for all five oral dose groups) with a higher percentage excreted in the urine of female rats. The majority of the urinary elimination (90

- 99%) occurred within the firs t 24 hours post

- dosing. A smaller percent (11

- 29% for all five oral dose groups) of the administered oral dose was eliminated in feces. The amount and rates of urinary and fecal elimination were comparable between XDE

729, administered as sodium salt (both labels) and XDE

- 729 methyl as a low oral dose. Rats orally administered 750 mg 14 C

- XDE

- 729 sodium salt/kg bw demonstrated decreased urinary elimination compared to rats administered 10 mg 14 C

- XDE

- 729 sodium salt/kg bw. There was no excretion of radioactiv ity via exhaled CO 2

. The IV

- administered XDE

- 729 sodium salt was also rapidly and extensively excreted in urine (85

- 88% of administered dose). The majority of the urinary elimination (98

- 99%) occurred in the first 24 hours post

- dosing. A smaller percent (11

- 15%) of the IV dose was eliminated in feces. At 168 hours post

- dosing, levels in the tissues ranged from non

- quantifiable to 0.3% of the orally administered dose from either XDE

- 729 (low single, high single, multiple dose, and low dose pyridine

- rin g label) or 14 C

- XDE

- 729 methyl (low single). Less than 0.03% of the IV administered 14 C

- XDE

- 729 remained in the tissues at 168 hours post

- dosing. In the rats orally administered as a single dose at 10 mg 14 C

- XDE

- 729 sodium salt/kg bw (both labels) or 1 0 mg 14 C

- XDE

- 729 methyl/kg bw, the C max was 1.6 to 3.6 fold lower for the methyl group. Elimination of the radioactivity from plasma was rapid during the initial distribution phase (t 1⁄2α = 0.5

- 0.9 hours) followed by a slower elimination during the terminal phase (t = 5

- 16 hours). Rats orally administered 750 mg 14 C

- XDE

- 729 sodium salt/kg bw displayed a slower initial distribution phase (t 1⁄2α = 2.7

- = 5.6

- 8.7 hours) was within the range of the 10 mg/kg bw group. Clearance of the IV

- administered XDE

- 729 sodium salt was ~ 0.5 mL/h/kg. The plasma AUC was 2.0

- 2.6 times higher at the high dose than would be expected for dose proportionality demonstrating a supraproportional increase in systemic exposure at the high dose. The AUC non

- linearity is consistent with the longer initial distribution phase of the 750 mg/kg bw group and decreased urinary elimination at 168 h post

dose, likely due to a slower redist ribution at the high dose and saturation of elimination. The time

- course of radioactivity in RBC was less than the plasma time

- course concentration profiles with lower concentrations. At 10 mg/kg bw oral dose (both labels, sodium salt and methyl forms) whe n compared to the concentrations in plasma, the maximum concentrations in RBC were 8

- to 33

- fold less. At the high dose of 750 mg/kg bw, the C max was 2

- to 3

- fold less than in plasma. The time to peak, maximum concentration and absorption half

- lives in RBC were comparable to plasma numbers. The time for distribution, as reflected by t , and elimination (t ) was less than those in plasma for all five treatment groups, with the exception of the high dose group which demonstrated a longer elimination (t ) i n RBC compared to plasma. The AUC numbersHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 115 of 126 were substantially less than those in plasma for all five treatment groups. Overall, similar kinetics were observed across sexes for the sodium salt and methyl test materials. The IV

administered XDE

- 729 sodium sal t demonstrated higher clearance values for RBC (5

- to 8

- fold) when compared to plasma. As seen in plasma in RBC’s there was a supraproportional increase in AUC from the low to the high dose, however, in contrast to plasma the AUC non

- linearity is consisten t with the longer terminal elimination phase of the 750 mg/kg bw group. Analysis of blood samples from the methyl dose group afforded primarily XDE

- 729 and no parent ester, indicating rapid hydrolysis of the ester in vivo . Although XDE

- 729 methyl showed in creased t max and decreased C max when compared to the same dose of XDE

- 729 sodium salt/kg bw, the administration of both test compounds resulted in similar AUC values. The major radiolabeled component present in the urine and fecal extracts from animals a dministered 14 C

- XDE

- 729 sodium salt, was XDE

- 729 acid. The major radiolabeled component present in urine and fecal extracts of animals dosed with XDE

- 729 methyl was the hydrolysis product, XDE

- 729 acid. Minor metabolites of both test materials consisted of the acyl

glucuronide conjugate of parent XDE

- 729 (present in selected samples at greater than 5% of the administered dose) and O

- demethylated XDE

- 729 and the corresponding sulfate and glucuronide conjugates. In summary, administered XDE

- 729 sodium salt o r methyl was rapidly and highly absorbed, poorly metabolized and readily excreted mainly in urine from the rat, with lesser amounts excreted in feces and very low tissue residues following single oral and IV low, high oral doses and low oral repeat doses. The similarity in absorption, tissue distribution, elimination kinetics and excretion, and metabolite profiles between XDE

- 729 sodium salt (low single, phenyl

- and pyridine

- ring labels) and XDE

- 729 methyl (low single phenyl

- ring label) and blood analyses s howing that the methyl ester is rapidly hydrolyzed to XDE

- 729 acid in vivo at 10 mg/kg bw , demonstrates bioequivalence between these two test materials at low doses. This study is classified as acceptable/guideline and satisfies the guideline requirement for a general metabolism study in the rat.
870.7600 Dermal Absorption
- Rat Study not required (see Executive Summaries for Halauxifen

- methyl)
A.4.9 Immunotoxicity
870.7800 Immunotoxicity Study conducted on halauxifen

- methyl

– see Executive Summaries for halauxifen

- methyl
A.4.9 Special/Other Studies
See Executive Summaries for halauxifen

- methyl METABOLITE X11449757 (GENOTOXICITY ONLY)Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 116 of 126 A.4.6 Mutagenicity
Gene Mutation Guideline 870.5100, Bacterial Reverse Mutation Test (Pre

- Incubation Assay) MRID 48557847 Acceptable/guideline 0, 156.25, 312.5, 625, 1250, 2500 or 5000 μg/plate

- S9 activation and 0, 51.2 , 1 28 , 32 0, 8 00, 2000 or 5000 μg/plate + S9 activation Negative in Salmonella typhimurium strains TA98, TA100 , TA102, TA1535, TA1537 for induced mutant colonies over background at the HGPRT locus up to precipitating and cytotoxic concentrations.
Guideline 870.5300, in vitro mammalian cell forward gene mutation CHO cells, HGPRT locus MRID 48557850 Acceptable/guid eline 0, 100, 200, 400, 800, 1600 or 3210 μg/ mL +/

- S9 activation Negative for induced mutant colonies over background at the HGPRT locus up to precipitating and cytotoxic concentrations.

Cytogenetics
Guideline 870.5375, in vitro mammalian cell chromosomal aberration assay, human peripheral lymphocytes MRID 00141673 Acceptable/guideline 0, 25.3, 50.6, 101.3, 202.5, 405 or 810 μg/ mL +/

- S9 activation Negative for increased chromosomal aberrations over background up to cytotoxic and precipitating concentrations.Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 117 of 126 A ppendix B
. Metabolism Summary Table s
Table B. 1 . Tabular Summary of Metabolites and Degradates Chemical Name Matrix Percent TRR (PPM) 1 Matrices

- Major Residue (>10%TRR) Matrices

- Minor Residue (<10%TRR)

Parent Halauxifen

Methyl Wheat Forage: PH 5.0 (0.010)

Wheat Forage: PY 8.3 (0.012) Wheat Hay: PH 1.0 (0.002) Wheat Hay: PY 5.4 (0.022) Wheat Straw: PH 1.2 (0.002) Wheat Straw: PY 1.9 (0.001) Turnip Tops: PH 13.5 (0.011) Turnip Tops: PY 12.2 (0.010) Rotational Crops

- - Ruminant Milk PY

- day 3: 0.4 (<0.001) Milk PY

- day 4: 0.5 (<0.001) Kidney PY: 0.6 (<0.001) Poultry Skin with Fat PH: 0.5 (<0.001) Rat <5% of the AD in excreta Water ND

- 97% Halauxifen

Methyl Glucose Conjugate Turnip Tops: PH 26.4 (0.021)

Turnip Tops: PY 28.7 (0.024) Rotational Crops

- - Ruminant

- - Poultry

- - Rat

- - Water

- - Halauxifen

Methyl N

- Glucuronic Acid Conjugate Rotational Crops

- -

Ruminant Milk PY

- day 3: 3.5 (<0.001) Milk PY

- day 4: 4.2 (<0.001) Poultry

- - Rat

- - Water

- - XDE

- 729 Acid Wheat Forage: PH 1.6 (0.003)

Wheat Forage: PY 1.9 (0.003) Wheat Hay: PH 1.7 (0.004) Wheat Hay: PY 1.8 (0.007) Wheat Straw: PH 1.2 (0.002) Wheat Straw: PY 1.8 (0.001) Turnip Tops: PH 3.1 (0.002)Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 118 of 126 Turnip Tops: PY 3.5 (0.003) Rotational Crops

- - Ruminant Kidney PY: 34.4 (0.014) Renal Fat PH: 44.8 (0.007) Liver PH: 12.1 (0.009) Kidney PH: 36.7 (0.044) Milk PY

- day 3: 2.0 (<0.001) Milk PY

- day 4: 3.6 (< 0.001) Liver PY: 5.8 (0.002)
Poultry Skin w/ Fat PH: 26.0 (0.044) Liver PH: 6.3 (0.003) Liver PY: 5.5 (0.002) Rat ND ND Water ND

- 99% XDE

- 729 Acid Glucose Conjugate Turnip Tops: PH 12.5 (0.010)

Turnip Tops: PY 12.6 (0.010) Rotational Crops

- - Ruminant

- - Poultry

- - Rat

- <5% of the AD in excreta Water

- - X11406790 Wheat Forage: PH 2.6 (0.005)

Wheat Forage: PY 3.8 (0.006) Wheat Hay: PH 0.5 (0.001) Wheat Hay: PY 0.8 (0.003) Wheat Straw: PH 0.9 (0.002) Wheat Straw: PY 0.8 (0.001) Turnip Tops: PH 2.2 (0.002) Turnip Tops: PY 1.5 (0.001) Rotational Crops

- - Ruminant Milk PY

- day 3: 2.5 (<0.001) Milk PY

- day 4: 0.3 (<0.001) Kidney PH: 1.2 (0.002) Kidney PY: 0.3 (<0.001) Liver PY: 8.8 (0.003) Poultry Skin with Fat PH: 4.3 (0.001) Liver PH: 5.7 (0.003) Rat

- - Water 0.5%

- 33% X11406790 Glucose Conjugate Wheat Forage: PH 2.6 (0.005)

Wheat Forage: PY 1.4 (0.002) Wheat Hay: PH 4.4 (0.010) Wheat Hay: PY 4.9 (0.020) Wheat Straw: PH 3.2 (0.006) Wheat Straw: PY 3.0 (0.002)Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 119 of 126 Rotational Crops

- - Ruminant

- - Poultry

- - Rat

- - Water

- - X11406790 Glucose Malonyl Conjugate Wheat Forage: PH 10.3 (0.021)

Wheat Forage: PY 5.9 (0.009) Wheat Hay: PH 5.0 (0.012) Wheat Hay: PY 4.8 (0.020) Wheat Straw: PH 5.2 (0.010) Wheat Straw: PY 3.7 (0.002) Turnip Tops: PH 5.9 (0.005) Turnip Tops: PY 7.2 (0.006) Rotational Crops

- - Ruminant

- - Poultry

- - Rat

- - Water

- - X11406790 Glucuronic Acid Conjugate Rotational Crops

- -

Ruminant Milk PY

- day 3: 40.7 (0.005) Milk PY

- day 4: 24.5 (0.002) Kidney PH: 5.9 (0.007)
Poultry

- - Rat

- - Water

- - X11406790 Sulfate Conjugate Rotational Crops

- -

Ruminant Milk PY

- day 3: 21.8 (0.003) Milk PY

- day 4: 12.5 (0.001) Kidney PH: 0.8 (0.001)
Poultry Skin with Fat PH: 12.3 (0.002) Liver PH: 14.5 (0.007) Liver PY: 8.3 (0.004)

Rat

- - Water

- - X11449757 Wheat Forage: PH 5.1 (0.010)

Wheat Forage: PY 4.7 (0.007) Wheat Hay: PH 1.9 (0.005) Wheat Hay: PY 1.9 (0.008) Wheat Straw: PH 2.0 (0.004) Wheat Straw: PY 1.7 (0.001) Rotational Crops

- - Ruminant Renal Fat PH: 17.4 (0.003) Liver PH: 62.0 (0.048)Halauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 120 of 126 Liver PY: 31.9 (0.010) Kidney PH: 14.7 (0.018) Kidney PY: 14.4 (0.006) Milk PY

- day 3: 10.1 (0.001) Milk PY

- day 4: 21.9 (0.002) Poultry Liver PH: 32.3 (0.015) Skin with Fat PH: 2.9 (<0.001) Rat <5% of the AD in excreta Water 3%

- 77% X11449757 Sulfate Conjugate Rotational Crops

- -

Ruminant Kidney PH: 18.3 (0.022) Kidney PY: 10.4 (0.004) Renal Fat PH: 9.5 (0.002)
Poultry Skin with Fat PH: 0.4 (<0.001) Liver PH: 0.9 (<0.001) Rat <5% of the AD in excreta Water

- - X11449757 Glucuronide Conjugate Rat

- <5% of the AD in excreta

Degradate 1 Water 20%

- 41% Degradate 2 Water 8%

- 13% Degradate 4 Water 0.7%

- 15% Degradate 10 Water ND

– 13% Degradate 11 Water ND

– 16% Degradate 14 Water ND

– 12% The wheat metabolism studies included use of a safener. The Turnip samples were taken at 14 days after treatment No metabolites were identified in the rotational crop study. TRR in all commodities was 0.001 ppm or less <5% of the AD: <5% of the administered dose

Table B.2. Structures of Halauxifen
- Methyl Metabolites and Degradates Code Name Chemical Abstracts Name Structure Where Found Halauxifen

methyl methyl 4

- amino

- 3

- chloro

- 6

- (4

-

chloro

- 2

- fluoro

- 3

- methoxyphenyl)pyridine

- 2

- carboxylate Metabolism: wheat, turnips, poultry, drinking water
Env fate: hydrolysis, soil photolysisHalauxifen

- methyl Human Health Risk Assessment DP No. D40 6145

Page 121 of 126 Table B.2. Structures of Halauxifen
- Methyl Metabolites and Degradates Code Name Chemical Abstracts Name Structure Where Found H alauxifen

methyl N

- glucose conjugate (anomers) methyl 3

- chloro

- 6

- (4

- chloro

- 2

-

fluoro

- 3

- methoxy

- phenyl)

- 4

- [[3,4,5

- trihydroxy

- 6

- (hydroxymethyl)tetrahydropyra n
- 2

- yl]amino]pyridine

- 2

- carboxylate (anomers = α

- and β

- con jugated glucose) Metabolism: turnips

H alauxifen

methyl N

- gluc uronic acid conjugate 6

- [[3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

3

- methoxy

- phenyl)

- 2

- methoxycarbonyl

- 4

- pyridyl]amino]

- 3,4,5

- trihydroxy

tetrahydropyran

- 2

- carboxylic acid

or isomer Metabolism: ruminants

XDE

- 729 Acid
4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

-

fluoro

- 3

- methoxy

- phenyl)

- pyridine

- 2

- carboxylic acid
Rat metabolism DER: XR

- 729 Parent Metabolism: wheat, turnips, ruminants, poultry, rats , drinking water
Env fate: hydrolysis, soil photolysis, anaerobic soil metabolismHalauxifen

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Page 122 of 126 Table B.2. Structures of Halauxifen
- Methyl Metabolites and Degradates Code Name Chemical Abstracts Name Structure Where Found XDE

- 729 Acid gl ucose conjugate [3,4,5

- trihydroxy

- 6

-

(hydroxymethyl)tetrahydropyra n
- 2

- yl] 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- methoxy

phenyl)pyridine

- 2

- carboxylate

or 3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

- methoxy

- phenyl)

- 4

- [[3,4,5

- trihydroxy

- 6

- (hydroxymethyl)tetrahydro pyra n
- 2

- yl]amino]pyridine

- 2

- carboxylic acid or Metabolism: turnips

X11406790 methyl 4

- amino

- 3

- chloro

- 6

- (4

-

chloro

- 2

- fluoro

- 3

- hydroxyphenyl)pyridine

- 2

- carboxylate Metabolism: wheat, turnips, ruminants, poultry, drinking water
X11406790 glucose conjugate Metabolism: wheat
X11406790 glucose

malonyl conjugate Metabolism: wheatHalauxifen

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Page 123 of 126 Table B.2. Structures of Halauxifen
- Methyl Metabolites and Degradates Code Name Chemical Abstracts Name Structure Where Found X11406790 N

- or O

glucuronic acid conjugate 6

- [[3

- chloro

- 6

- (4

- chloro

- 2

- fluoro

- 3

-

hydroxy

- phenyl)

- 2

- methoxycarbonyl

4

- pyridyl]amino]

- 3,4,5

- trihydroxy

tetrahydropyran

- 2

- carboxylic acid or 6

- [3

- (4

- amino

- 5

- chloro

- 6

- methoxycarbonyl

- 2

- pyridyl)

- 6

- chloro

2

- fluoro

- phenoxy]

- 3,4,5

- trihydroxy

tetrahydropyran

- 2

- carboxylic acid or Metabolism: ruminants

X11449757 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

-

fluoro

- 3

- hydroxyphenyl)

- pyridine

2

- carboxylic acid
Rat metabolism DER: O

demethyl

- XR

- 729 Metabolism: wheat, ruminants, poultry, rats drinking water
Env. fate: anaerobic soil metab., aerobic aquatic metab.
X11449757 Sulfate Conjugate 4

- amino

- 3

- chloro

- 6

- (4

- chloro

- 2

-

fluoro

- 3

- sulfoxy

- phenyl)pyridine

2

- carboxylic acid
Rat metabolism DER: sulfate conjugate of O

- demethyl

- XR

- 729 Metabolism: ruminants, poultry, rats

X11449757 Glucuronide Conjugate Rat metabolism DER: glucuronide

conjugate of O

- demethyl

- XR

- 729 Metabolism: rats
X11449757 Acyl Glucuronide Conjugate Rat metabolism DER: acyl

glucuronide conjugate of O

demethyl

- XR

- 729 Metabolism: ratsHalauxifen

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Page 124 of 126 Table B.2. Structures of Halauxifen
- Methyl Metabolites and Degradates Code Name Chemical Abstracts Name Structure Where Found Degradate 1 Unknown

Unknown Metabolism: drinking water
Env. fate: aqueous photolysis Degradate 2 Unknown

Unknown Metabolism: drinking water
Env. fate: aqueous photolysis Degradate 4 Unknown

Unknown Metabolism: drinking water
Env. fate: aqueous photolysis Degradate 10 Methyl 4

- amino

- 6

- (4

- chloro

- 2

-

fluoro

- 3

- methoxy

- phenyl)

- 3

- hydroxy

- pyridine

- 2

- carboxylate Metabolism: drinking water
Env. fate: aqueous photolysis Degradate 11 Methyl (2E)

- 2

- (4

- chloro

- 2

- fluoro

3

- methoxy

- benzoyl)iminoacetate Metabolism: drinking water
Env. fate: aqueous photolysis Degradate 14 2

- [(E)

- (4

- chloro

- 2

- fluoro

- 3

-

methoxy

- phenyl)methyleneamino] acetaldehyde Metabolism: drinking water
Env. fate: aqueous photolysisHalauxifen

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Page 125 of 126 Appendix C
. Physical/Chemical Properties

Table C
- 1 . Physicochemical Properties of the Technical Grade Test Compound

Parameter Value for Halauxifen

- methyl Value for XDE

- 729 Acid (X11393729) Reference Water solubility pH 5 pH 7 pH 9 unbuffered mg/L at 20°C 1.66 1.67 1.69 1.83 at 20°C 102 mg/L 3.07 g/L 46.1 g/L 84.0 mg/L Tolerance

Petition 2E8086

Octanol/water partition coefficient at 20°C Log K OW Log K OW pH 5 buffer solution 3.75 0.42 pH 7 buffer solution 3.76

- 0.83 pH 9 buffer solution 3.92

- 1.01 Vapor Pressure 5.9 x 10

- 9 Pa at 20°C (4.4 x 10

- 11 mmHg) 1.5 x 10

- 8 Pa at 25°C (1.1 x 10

- 10 mmHg) 2 x 10

- 5 Pa at 20°C (1.5 x 10

- 7 mmHg) 3 x 10

- 5 Pa at 25°C (2.3 x 10

- 7 mmHg) Organic solvent solubility g/L at 20°C g/L at 20°C methanol 38.1 4.2 acetone >250 4.4 ethyl acetate 129 1.2 n
- octanol 9.83 0.43 Melting point 146°C not available pH 6.24 not available Dissociation constant (pK a ) 2.84 @ 20°C 5.11 @ 21.9°CHalauxifen

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Page 126 of 126 Appendix D
. Review of Human Research
This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical

. These data, which include studies from PHED 1.1 and the AHETF database are (1) subject to ethics review pursuant to 40 CFR 26, (2) have received that review, and (3) are compliant with applicable ethics requirements

. For certain studies, the ethics review may have included review by the Human Studies Review Board

. Descriptions of data sources, as well as guidance on t heir use, can be found at the Agency website 1

.

1 http://www.epa.gov/pesticides/science/handler

- exposure
- data.html and http://www.epa.gov/pesticides/science/post

- app
- exposure
- data.html